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5th Biennial Conference on Heart Valve Biology and Tissue Engineering
- Conference date: 18-20 May 2012
- Location: Mykonos Island, Greece
- Volume number: 2012
- Published: 01 May 2012
1 - 50 of 86 results
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Regulation of Abnormal Atrioventricular Valve Development
Authors: Huseyin C. Yalcin, Sarah Ajaeb and Jonathan T. ButcherAbstractHemodynamic forces play an essential role in driving the morphogenesis of the embryonic heart and its valves. We previously found that genes that regulate heart morphogenesis like BMP2 and VEGFA, have hemodynamically specific patternings. However, how mechanical signals like wall shear stress (WSS) affect these expression levels to regulate heart morphogenesis is not fully understood. A detailed understanding of the hemodynamic environment and gene expression patterns inside normally and abnormally growing embryonic hearts would help inform the clinical progression of congenital heart defects and develop treatment strategies to restore these defects. We previously developed a system to quantify local fluid forces within growing embryonic heart where we combined imaging modalities with computational fluid dynamics (CFD), and quantified the hemodynamics within the AV canal and OFT in chicks. In the current study, we analyzed how altered hemodynamics drives changes in local gene expression and downstream morphogenesis of AV valves. Perturbed blood flow was created via either left atrial ligation (LAL) at HH24 or right atrial ligation (RAL) at HH25, to constrict blood flow on respective side of the heart. Hemodynamic environment in the AV canal for operated embryos were compared to control groups via doppler ultrasound at HH31. Gene expression levels were analyzed using RT-PCR. Heart morphology was analyzed at HH31 via micro-CT and histology. RT-PCR results at HH25 showed that LAL caused a drastic decrease in gene expression levels in LV myocardium (VEGFA expression 18.2±5.6%, BMP2 expression 21.7±1.4% compared to controls) and in AV cushions (VEGFA expression 9.8±8.1%, BMP2 expression 3.5±1.9 %), whereas in RV myocardium, expression levels were not affected significantly (VEGFA expression 77.5±5.8% , BMP2 expression 66.0±10.0%). Doppler ultrasound showed that neither peak blood velocities nor time averaged velocities in right and left AV canal are different for both LAL and RAL at HH31, suggesting WSS levels are not different between experimental groups (max shear stress 287dynes/cm2) at that stage. For LAL embryos, left AV valve area was smaller (48±4% vs. 67±6%, p<0.05, valve areas normalized to total left and right AV valve areas) and right valve area was bigger (52±4% vs. 33±6%, p<0.05) compared to controls, whereas in RAL embryos, opposite was true at HH31, right AV valve are was smaller (12±5 % vs. 33±6%, p<0.05) and left AV valve area was larger (88±5% vs %67±6, p<0.05). The ratio of right ventricle to left ventricle was significantly larger in LAL embryos compared to RAL embryos (1.1 vs 0.55). Our results show that morphological abnormalities follow the alterations in gene expression levels which are caused by changes in mechanical signals due to altered hemodynamics. We found that at later stages hemodynamics is restored to keep the blood circulation normal, with an altered morphology.
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Contribution of epicardially derived cells to the leaflets of the murine AV valves
Authors: John Burch, Maurice Van den Hoff, Marie Lockhart, Laura Briggs and Andy WesselsAbstractTo determine the spatiotemporal contribution of epicardially derived cells (EPDCs) to the leaflets of the developing atrioventricular (AV) valves in the murine heart we have used a mWt1/IRES/GFP-Cre mouse and traced the fate of EPDCs from embryonic day (ED)10 until birth. Migration of EPDCs into the mesenchyme of the AV cushions starts around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets that derive from the lateral atrioventricular cushions, i.e. the mural leaflet of the left AV valve and the lateral leaflet of the right AV valve. In these developing leaflets the EPDCs eventually largely replace the endocardially-derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the respective leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in heart development.
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Matrix-controlled In-growth Selectivity as a Principle of Heart Valve Regeneration
Authors: Neil Davies, Mona Bracher, Michelle Sun, Deon Bezuidenhout and Peter ZillaAbstractSpontaneous scaffold based tissue regeneration may be a promising alternative to in vitro tissue engineered heart valve prostheses. Selective promotion of ingrowth of desired cells versus suppression of potentially harmful cell invasion would be a key principle of such an approach. We have begun to demonstrate the utility of hydrogels engineered with specific adhesive and degradation peptide sites towards achieving this long-term goal. Polyethylene glycol hydrogels that through Michaels addition chemistry allow for both polymerization via enzymatically (matrix metalloproteinase (MMP)) degradable peptides and appendage of adhesive peptides were developed. Hydrogels were crosslinked with either a relatively enzymatically promiscuous peptide (MMP-pep) or sequences specifically degradable by either MMP-14 or MMP-9 (MMP-14pep, MMP-9pep). MMPs are of interest as there is evidence of cell specific expression. For example in vascular cells under physiological conditions MMP-9 is expressed at low levels except in macrophages. Adhesive peptides utilised were RGD, YIGSR and/or PHRSN. A range of primary vascular cells (smooth muscle cells (SMC), endothelial cells (EC), valvular intestitial cells (VIC) and fibroblasts (FB)) were cultured individually or as spheroids in 2 or 3-D. Migration and invasion were then assessed by time-lapse phase and confocal microscopy. ECs were shown to migrate significantly more rapidly on a surface containing YIGSR/RGD relative to other peptide combinations whilst the migration of SMC was unaffected. Hydrogels polymerized with MMP-14pep or MMP-9pep were shown to be highly preferentially cleaved by their relative enzymes. VICs, Fb and SMC primarily utilised MMPs to invade the hydrogels as demonstrated by inhibition of sprout formation by the MMP inhibitor, GM6001. VICs and FB invaded the gels in the following order, MMP-pep>>MMP-14>MMP-9. However, SMC showed a marked preference for invasion into MMP-14pep crosslinked hydrogels above that observed in their MMP-pep and MMP-9pep counterparts. Confocal microscopy analysis also showed a significantly more branched and interconnected pattern of invasion for SMC in MMP-14pep hydrogels. Thus we have begun to show that through engineering of extracellular matrix mimics with precise enzymatic and adhesive recognition sites it is possible to selectively influence specific cell types invasive behaviour suggesting that cell-specific ingrowth scaffolds may be achievable.
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Investigating the Contribution of Pathological Levels of Cyclic Strain on Vascular and Valvular Calcification
Authors: Zannatul Ferdous, Hanjoong Jo and Robert NeremAbstractAortic stenosis and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of calcification are still not clearly understood. To understand the contributions of physiological (10%) and pathological (5%, 15%) levels of cyclic strain in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells (HASMC) and human aortic valvular interstitial cells (HAVIC), both isolated from non-calcific heart transplant tissues. The tissue engineered collagen gels were cultured in standard osteogenic media for three weeks in a custom designed bioreactor and all assessments were performed at the end of the culture period. The major finding of this study was that bone morphogenic protein (BMP) -2, -4 and transforming growth factor (TGF)-β1 mRNA expression significantly changed in response to the magnitude of strain in valvular cells, while the least expression was observed for the representative 10% physiological strain. On the other hand, these mRNA expressions in vascular cells responded to strain, but did not vary due to the magnitude (5% versus 10% versus 15%) of strain. When the BMP-2 and BMP-4 protein expression was detected using immunostaining, we observed that only valvular cells showed greater BMP-2 expression for 5% and 15% strain when compared to 10% strain within the same cell type. Our results suggest that cell mediated differences exist between vascular and valvular cells in their response to different levels of cyclic strain.
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The Role of Integrins on the Regulation of Contractility in Mitral Valve
Authors: Marzieh Zamani, Padmini Sarathchandra, Najma Latif, Magdi H. Yacoub and Adrian H. ChesterAbstractThe mechanical properties of heart valves are modulated by the contractile response of valve interstitial cells (VICs). It is unknown how the contractile responses of VICs are translated to the extra cellular matrix to alter the stiffness of the valve tissue. We have studied the signalling mechanisms of integrin subunits and specifically α2β1 integrin, a predominant collagen binding molecule, in mediating the contractility of porcine mitral valve tissue and cells. The expression of integrins was analysed by immunostaining of cells. The contractility of porcine mitral VICs in the presence and absence of a α2β1 integrin blocking antibody and the effect of different signalling inhibitors was analysed in a collagen gel. The effect of modulators of contractility was determined on mitral valve tissue incubated in organ baths which challenged with endothelin-1 (10-10-10-7M) following 24 hours incubation with α2β1 integrin blocking antibody. Immunohistochemistry confirmed positive staining for α2β1 integrin and the expression of α1, α2, α3 and β1 in the VICs. The α2β1 blocking antibody was able to significantly reduce the contraction of collagen gels from 63.6±7.8 to 23.4±4.3 % (p<0.001) of control (no cells in gel). PF573228, the inhibitor of focal adhesion kinase, caused a reduction of gel contraction by 30% of the control. The maximum contractile response of cusp tissue to ET-1 maintained under isometric conditions in tissue baths was significantly reduced from 9.5±5.0 to 3.0±2.5mN/g (p<0.001) wet weight by the presence of the α2β1 integrin blocking antibody. These results demonstrate a role for α2β1 integrin in linking the contractile response of valve cells to the extra cellular matrix and highlight further the complex interactions between the cells and the extra cellular matrix of the valve.
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Modeling Flow-Responsive Gene Regulation In Native Valve Tissue
Authors: Arpi Siyahian, Fariba Chalajour, Xiaoyuan Ma, Frank L. Hanley and R. Kirk RiemerAbstractA variety of approaches to heart valve tissue engineering are underway by many groups of investigators. We believe that a thorough understanding of the biophysical and biomolecular interactions that mediate normal leaflet homeostasis will inform improved approaches to the engineering of truly regenerative heart valve grafts. However, our understanding of the fundamental biology of normal valve tissue is still quite limited. This informational gap has led us to develop an ex vivo system for long-term valve culture in order to study the mechanobiology of native tissue. Using this system, we have cultured trileaflet rat heart valves with and without flow-induced valve cycling. We report here the preliminary findings about the effects of both ex vivo culture and of valve cycling on pulmonary leaflet cell gene expression following 7 days of flow culture. Expression of leaflet mRNA was queried using Affymetrix whole genome microarrays and the results analyzed using GeneSpring, EXPLAIN and MetaCore (GeneGO) software. Our analysis revealed 2147 genes whose expression was altered under flow conditions, and 1918 genes under static conditions. 1501 genes were common to both conditions. The effects of culture on gene expression affected multiple pathways, but the most active gene groups involved pathways for cytoskeletal remodeling, development, and cell adhesion. The genes involved in these pathways were altered in both culture conditions. Changes in cytoskeletal and ECM remodeling genes were more prominent in the static condition, while those involved in VEGF signaling were seen under flow condition. Our previously reported histological analyses of cultured rat pulmonary valve leaflet tissue revealed that flow promotes the maintenance of tissue integrity while the lack of flow leads to extracellular matrix remodeling and fibrinoid tissue formation. These gene expression analyses confirm the expected role of flow in maintaining leaflet tissue integrity as evidenced by its induction of VEGF signaling genes, while the lack of flow induces changes in genes involved in extracellular matrix and cytoskeletal remodeling.
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Inflammation Drives Endothelial-to-Mesenchymal Transition and Interstitial Calcification in 3D in Vitro Culture
Authors: Jennifer Richards, Emily Farrar, Gretchen Mahler and Jonathan ButcherAbstractCalcific aortic stenosis is a serious pathology that accounts for 43% of those suffering from heart valve disease. Valve disease initially presents with inflammation and endothelial dysfunction, which can lead to an endothelial-to-mesenchymal (EndMT) shift in the endothelial layer of the valve. It is not yet well understood how valve endothelial cells (VEC) regulate valve interstitial cells (VIC) in inflammatory or osteogenic environments. We here present a 3D culture system that models cellular responses and interactions when exposed to different environmental conditions. Porcine aortic VEC and/or VIC were cultured in mechanically constrained 3D type I collagen hydrogels for up to 14 days. Early disease studies introduced different dosages of TNFα to VEC on gel surfaces for up to 48 hours. EndMT was assessed via real time PCR and cell invasion within the matrix. Apoptosis and proliferation in VEC via TUNEL and anti-BrdU IHC staining were also evaluated in early inflammatory conditions. Later early-stage disease studies introduced osteogenic and inflammatory environments to 3D VIC/VEC gels through the addition of osteogenic differentiation factors into the culture media (OGM), or 30 ng/ml TNFα. Calcium deposition within the matrix was measured via Alizarin Red staining. Real time PCR was used to evaluate the expression of calcification-related genes such as osteocalcin and runx-2. Early inflammatory conditions as induced by addition of TNFα for 48 hours stimulated EndMT-like VEC activation, including matrix invasion and upregulation of αSMA and snail. Addition of TNFα increases VEC proliferation in a dose dependent manner. TNFα does not significantly increase apoptosis, even at the highest dosage levels. VIC gels cultured in OGM for 14 days significantly calcified and expressed osteocalcin and runx-2. Co-culture with VEC inhibited both matrix calcium deposition and osteoblastic differentiation. When 30 ng/ml TNFα was added to VIC 3D cultures, TNFa induced increased matrix calcium deposition in 14 days, which was then mitigated by co-culture with VEC. The inhibitory effect of VEC on VIC calcification in OGM was blocked when TNFα was introduced to the culture. These results demonstrate that inflammatory microenvironments induce calcification in valve interstitial cells. There is a protective role for the valve endothelium in VIC calcification, which could be inhibited by EndMT induced by the inflammatory conditions found in diseased valves. We found that apoptosis is not a driving factor in inflammatory activation of VEC or osteogenic differentiation of VIC. Targeting TNFα and EndMT signaling could be important therapeutic strategies. More generally, targeting VEC is a promising approach for early treatment of valve disease. This co-culture 3D system is a powerful tool to elucidate mechanisms of valve disease.
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Atypical expression and regulation of smooth muscle markers in human calcified valves
Authors: Najma Latif, Padmini Sarathchandra, Magdi H. Yacoub and Adrian H. ChesterAbstractCalcific aortic stenosis and atherosclerosis share many similar characteristics suggesting that similar mediators and pathogenic pathways are involved. In both conditions, the native cells are quiescent. However upon damage, both valve interstitial cells (VICs) and smooth muscle cells (SMCs) in the valve and the vascular wall respectively undergo remodelling and transformation. We hypothesise that the smooth muscle cell phenotype plays a role during the calcification process. 12 normal and 16 calcified valves were analysed for smooth muscle markers including smooth muscle α-actin, smooth muscle myosin, calponin, caldesmon, desmin, SM1 and SM2 by immunocytochemistry. The expression of myocardin-related transcription factors, (MRTFA/B) as key regulators of smooth muscle gene expression, was also evaluated. Normal human valves demonstrated the expression of smooth muscle markers localised to the base of the valve. Occasionally smooth muscle markers were seen in small groups of cells in the region from the base to the central region of the valve but never in the region from the central to the co-apting edge. Smooth muscle markers in all the calcified valves demonstrated an increased and aberrant expression. The expression of smooth muscle myosin, SM1, SM2, calponin and smooth muscle α-actin was found to be abundantly expressed around calcified nodules and distal to the nodules. The pattern of staining was frequently localised along the majority of the valve tissue as well as in isolated cells. Additionally, regions of the endothelium and endothelial cells lining the vessels were found to be positive for smooth muscle markers in 9/16 calcified valves. Normal valves did not demonstrate any expression of MRTFs. However, MRTF-A and MRTF-B were induced in calcified valves in a similar pattern to the expression of smooth muscle markers. They were also induced in the endothelium and the endothelial cells lining some vessels. Importantly, the expression of MRTFs was nuclear in both VICs and endothelial cells indicative of activation. TGFβ1 (10ng/ml) was able to induce and translocate the expression of MRTF-A to the nucleus in VICs within 4 hours in some isolates. Calcified valves harbour a greatly increased number of smooth muscle marker-positive VICs and endothelial cells. This aberrant expression in endothelial cells may emulate endothelial to mesenchymal transformation (EMT) during embyonic valvulogenesis. Concomittantly, calcified valves expressed MRTFs, key regulators of smooth muscle gene expression. The presence of smooth muscle cell markers and MRTFs in calcified valves suggest a role for this cell phenotype in the development of the calcification process.
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Characterization of Early Porcine Aortic Valve Disease
Authors: Krista L. Sider, Andrea V. Kwong, Bowen Wei and Craig A. SimmonsAbstractIn calcific aortic valve disease (CAVD), pathological differentiation of valve interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. We have shown that VIC pathological differentiation in vitro is sensitive to matrix stiffness. We therefore hypothesized that focal changes in the stiffness of the fibrosa with CAVD progression correlate with alterations in the extracellular matrix (ECM) and pathological cell phenotypes in vivo. Twenty-four male Yorkshire pigs were fed either a control or experimental diet supplemented with 12% lard and 1.5% cholesterol for 2 or 5 months (n = 6 per group). Effective moduli were measured in focal regions of the fibrosa (n = 295) and ventricularis (n = 142) layers of intact right coronary aortic valve leaflets by micropipette aspiration. Histology was conducted within radial center sections and selected focal test locations within the fibrosa from each leaflet to characterize local ECM and cell phenotypes. Early stage disease was induced by the experimental diet, as evidenced by more significant proteoglycan-rich lesions (~10 - 200 µm thick) onlayed on the fibrosa (p < 0.05, controlling for genetic variability). Interestingly, lipid deposition was seen in only 28% of regions with early lesions, and 15% of regions without early lesions. No osteoblastic or macrophage cells were present in control or experimental valves, with only slight myofibroblast presence at the base of some 5 month lesions. Chondrogenic Sox9-positive cells were observed extensively and were positively correlated with proteoglycan content (p < 0.0001). Regions within the fibrosa were significantly stiffer than those in the ventricularis in both control and experimental valves (p <0.001). Although there were no significant differences between the control and experimental overall tissue moduli at these early time points, modulus heterogeneity did increase with disease. Furthermore, lesions in the fibrosa had lower moduli than non-lesion fibrosa locations (p < 0.06). These soft lesions contained significantly more proteoglycan (p <0.001) and Sox9 expression (p = 0.014) than normal fibrosa tissue. In conclusion, ECM remodelling can occur in a porcine model of early CAVD in the absence of lipid deposition, inflammatory cells, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich lesion and chondrogenic cell presence. Mechanically soft proteoglycan-rich lesions may support chondrogenic VIC differentiation, providing new insights into early CAVD pathogenesis.
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The role of Insulin-like Growth Factor-1 in Calcification of Human Aortic Valves
AbstractInsulin-like Growth Factor-1 (IGF-1) is a multi-functional protein that plays a role in survival, growth, proliferation and differentiation of various cell types. Particularly in the bone, IGF-1 is involved in tissue formation and growth, and the lack of expression of the gene leads to striking features such as short bone phenotype and low bone mineral density. In atherosclerosis, IGF system has been shown to stimulate vascular smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) synthesis contributing to maintenance of plaque stability. Consequently, a reduction of IGF-1 in atherosclerotic plaques has been suggested to increase SMC apoptosis and reduce ECM synthesis leading to weakening of the plaque. Despite extensive studies on its role in various organs and disease models, there is very little known about the effects of IGF-1 in human aortic valve (hAV) tissue that can suffer damaging effects such as cell death, differentiation and tissue mineralization, resulting into Aortic Valve Stenosis (AVS). The objective of the present study is therefore to investigate potential function of IGF-1 in this disease model. Human aortic valve samples were collected from patients undergoing aortic valve replacement or heart transplantation. The tissue expression of IGF-1 was analysed at mRNA level by quantitative Realtime PCR in calcified hAV (n=10-14) and non-calcified hAV (n=3-6), using the comparative Ct method. In vitro, the expression of endogenous IGF-1 was investigated on human valve interstitial cells (hVICs) upon administration of calcifying medium with or without transforming growth factor-beta1 (TGF-b1), a known pro-mineralization molecule (n=3). In addition to the expression of IGF-1, alkaline phosphatase (ALPL), TGF-b1 and osteopontin (OPN) were also examined at RNA level. IGF-1 expression was significantly up-regulated in the diseased valves compared to the controls (4.10 +/- 1.20 calcified vs. 1.10 +/- 0.76 control, p=0.0127). In vitro, there was an up-regulation of IGF-1 (p=0.06) and TGF-b1 expression (p=0.02) upon TGF-b1 stimulation. OPN expression was also highly up-regulated in calcifying media treated cultures, independent of TGF-b1 treatment. ALPL expression in contrast, was higher in normally growing cells compared to TGF-b1 and/or calcifying media treated cells (p=0.05). The significant elevation of IGF-1 in calcified valves in this small patient cohort strongly suggests that IGF-1 might be a key player in AVS disease. Whether the elevated IGF-1 expression is an effect or the cause of calcification is currently under investigation. Our preliminary data on TGF-b1 treatment of hVICs suggests that IGF-1 expression might be an effect of cells to counter apoptotic death conferred by TGF-b1 treatment. Further work on the detailed molecular mechanisms involved in IGF-1 action and its downstream signalling pathways is needed.
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Reversible Effects of Polyunsaturated Fatty Acids on Activation of Interstitial Cells From Porcine Aortic Valves
Authors: Wolfgang Witt, Anett Jannasch, Klaus Matschke and Thomas WaldowAbstractValvular interstitial cells (VICs) constitute the most prominent cell type in aortic valve cusps. VICs are a heterogeneous group of cells of multiple origins with mostly fibroblast properties. Like fibroblasts in general, VICs can acquire an activated state (myofibroblasts) upon exposure to several stimuli including mediators of inflammation, growth factors, mechanical stress or changes of ECM composition. An increase of activated VICs in adult valves is considered as indicator of pathological developments. Cultivation of VICs using standard 2D cell culture procedures also induces VIC activation resulting in contraction and spontaneous nodule formation. Our previous work has shown that this phenotype switch can be reversed by exposure to polyunsaturated fatty acids (PUFAs). In order to investigate underlying cellular mechanisms of the PUFA effects, VICs from porcine aortic valves were isolated and subcultured on collagen-coated surfaces. Spontaneous nodule formation after transfer to uncoated polystyrene was completely blocked by docosahexaenoic acid (DHA) and arachidoic acid (ARA) whereas eicosapentaenoic acid and a commercial extract from fish oil (Omacor) were less active. Oleic acid and palmitic acid were without effect. Treatment with ARA or DHA reduced the expression of myofibroblast marker proteins, α-smooth muscle actin (SMA) and myosin II, of a key enzyme of collagen synthesis (prolyl 4-hydroxylase), and of the phosphorylated (inactive) form of the F-actin severing protein, cofilin. In contrast, the abundance of fibroblast marker S100A4 was increased after treatment with PUFAs. The steady state level of active RhoA was reduced in the presence of DHA and ARA, and inhibition of RhoA or ROCK elicited the same effects as PUFAs. Finally, exposure to ARA and DHA reduced the G/F-actin ratio, and stabilization of F-actin with jasplakinolide blocked the effect of PUFAs on the expression of myofibroblast markers and on nodule formation. After culturing VICs with PUFAs for 14 days and subsequently in the absence of PUFAs for 4 days, cells regained the myofibroblast phenotype, showing the PUFA-induced phenotype switch was fully reversible. In conclusion, the results suggest that the differentiation of VICs to myofibroblasts can be truly reversed by certain PUFAs via the RhoA – ROCK – G-actin pathway, whereas an alternative mechanism, the preferential outgrowth of a undifferentiated subpopulation of VICs, seems to be unlikely since the PUFA effect was fully reversible.
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Pentraxin 3 is a Potential Diagnostic Marker for Aortic Stenosis
AbstractPentraxin 3 (ptx3) is a member of the long pentraxin family and is rapidly produced and released by many cell types, including endothelial cells, in response to primary inflammatory signals. In our preliminary work, PTX3 gene expression was significantly increased in aortic valve tissue following exposure to elevated cyclic pressure. Consequently, we hypothesized that ptx3 would be a useful biomarker for the early diagnosis of aortic valve sclerosis. Isolated aortic VICs were treated with Angiotensin II (300nM), TNF-alpha (10 ng/ml) or elevated cyclic pressure for six hours. The cell culture supernatant was collected and used to determine ptx3 protein expression using ELISA. Total RNA was isolated from the cells and PTX3 gene expression was determined using semi-quantitative RT-PCR. In addition to cell culture studies, ptx3 protein expression was determined in hypertensive New Zealand White rabbits. Rabbits underwent Goldblatt one-clip/one-kidney surgery to induce hypertension (n=5). Four sham models served as a control. Blood pressure, echocardiography data and serum samples were collected at 0, 2 and 4 months. After 4 months, rabbits were euthanized and aortic valve tissue was collected for histological and gene expression analysis. Data from the cell culture studies showed that elevated cyclic pressure caused an increase in PTX3 gene expression and ptx3 protein expression. However, no significant changes were observed in gene or protein expression from cells treated with Ang II or TNF-alpha. Data collected from the animal model showed that blood pressure increased significantly for the experimental group but not the control group. Levels of ptx3 protein were measured from the serum and showed a slight increase over the four-month course of the experiments. The data suggest that ptx3 expression is mechanosensitive in the aortic valve and is not stimulated by biochemical factors such as TNF-alpha or Ang II. The increase in ptx3 expression in hypertensive rabbits demonstrates that this could be a potential diagnostic marker for aortic stenosis.
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Effects of Finite Deformation on Extracellular Matrix Production and Mechanical Properties
AbstractWith our ability to incorporate viable cells distributed throughout the scaffold, we are provided a unique, controllable platform to develop a generalized finite deformation framework than can be used to gain an understanding of how the evolving extracellular matrix phase contributes to the construct gross mechanical behavior. The biosynthetic response of microintegrated VSMC’s was investigated low (15%), intermediate (30%), and high (50%) strain groups. These magnitudes were chosen as they correspond to a wide range of NAR deformations and physiologically relevant. A constant, quasi-static strain rate as applied sufficient to obtain a 1 Hz cycle duration. Culture durations of 7, 14, and 21 day time points were used for each strain level to quantify the ECM synthesis capacity of VSMC microintegrated in electrospun PEUU. A static group was also preformed at each time period. Results indicate that VSMC biosynthetic behavior is function of global strain with peaked soluble collagen synthesis was observed in specimens exposed to 30% strain. Our primary goal was to elucidate the mechanical behavior characteristics of the de-novo formed ECM. We determined the matrix mechanical contribution by assuming that the total mechanical response is simply the summation of the individual phases and any potential interactions that might arise between them. We thus quantified the collagen mass fraction and utilized an enzymatic technique to remove the ECM from the constructs, then retested them to obtain the degraded scaffold only responses. Results indicated that the newly formed matrix phase exhibit a highly anisotropic biaxial response, and was over 100 fold stiffer than similar ECM formed using stiff scaffolds previously studied in our lab. Moreover, the formed ECM had predicted mechanical properties similar to glutaraldehyde treated pericardium, a common heart valve biomaterial. Interestingly, peak biosynthetic activity correlates well with in vitro principle strain levels observed in PEUU tri-leaflet valves exposed to native ovine right side pressure. This suggests that physiologic hemodynamic conditions are optimal for the development of robust ECM accretion. To our knowledge, this is the first reported study to consider the effects of large deformation and the corresponding outcomes in terms of ECM mechanical integrity. Furthermore, the results reveal interesting insights into the functional role of the matrix accretion process in engineered tissues.
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Bio-inspired Anisotropic Nanofibrillar Matrices for Heart Valve Engineering
Authors: Jerome Sohier, Ivan Carubelli, Najma Latif, Padmini Sarathchandra, Adrian H. Chester and Magdi YacoubAbstractOur strategy for heart valve tissue engineering is the use of autologous cells to populate appropriate template matrices. In this context, an important goal is to devise a suitable biomimetic scaffold that supports proper cell growth and cell-matrix interactions by reproducing the specific anisotropic fibrillar structure of valves extracellular matrix (ECM). A novel type of highly porous anisotropic nanobrillar matrices was developed and evaluated with regards to structure, mechanical properties and ability to support human adipose derived stem cell (hADSC) colonization, growth and ECM production in vitro. Nanofibrillar structures were obtained by jet-spraying poly (ε-caprolactone) dissolved in chloroform on a variably rotating drum. Morphological evaluations of the structures were performed using scanning electron microscopy while porosity was calculated from polymer density, weight and volume. Elastic modulus of dry scaffolds (10x6x1 mm, n=5) was measured with a planar biaxial test bench with displacement rate of 0.05 mm/s. Human adipose derived stem cells (500,000) were top and rotary seeded on nanofibrillar discs (diameter 1 cm and thickness 0.8 mm) and cultured in 10 ml of complete medium under rotation (10 rpm) for 18 days. Histology (DAPI staining), DNA quantification and immunohistochemistry were used to characterize the resulting cellularized structures. The speed of drum rotation was adjusted up to 3000 rpm to produce highly aligned fibres (600 nm of average diameter) from the sprayed polymer. In conjunction with fibres anisotropy, the scaffolds Young's modulus was simultaneously increased (from 0.3 to 0.7 Mpa) and decreased (from 0.3 to 0.01 Mpa) longitudinally and orthogonally to fibre alignment, respectively. In addition, fibre alignment further increased scaffolds porosity from 97 % (isotropic) to 99%. Anisotropic matrices allowed a more extensive cellular invasion than isotropic scaffolds, possibly linked to their higher porosity and therefore open structure. hADSC proliferated significantly (up to 6-fold DNA increase), bridged the entire scaffolds thicknesses after 10 days and produced their own ECM as evidenced by collagen I production. This study highlights the potential of a newly developed highly porous anisotropic nanofibrillar matrices as substrate for tissue engineering, and in particular for heart valve engineering.
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PCL Scaffolds and Reduced In-Vitro Cell Expansion to Improve Engineered Valvular Tissue Formation
AbstractDifferent types of synthetic scaffolds are used for tissue engineering of heart valves. Tissue-engineered heart valves (TEHV), based on a rapid degrading polyglycolic acid (PGA) scaffold coated with poly-4-hydroxybutyrate (P4HB) and seeded with vascular derived cells, have shown promising in-vivo results. However, a major drawback of these TEHV is compaction and retraction of the leaflets causing regurgitation. It is hypothesized that this is a result of traction forces exerted by the cells, combined with an imbalance of the formed tissue and loss of mechanical integrity of the scaffold due to degradation. The aim of this study is to evaluate alternative approaches to overcome the compaction and retraction of TEHV without compromising on tissue composition and properties. The alternative approaches that are studied here are 1) the use of the slow degrading poly-ε-caprolactone (PCL) scaffold for prolonged mechanical integrity and 2) the use of lower passage vascular cells for enhanced tissue formation. Compaction, tissue formation, cell phenotype and mechanical properties of tissues based on passage 3, 5 and 7 vascular cells in PCL and PGA-P4HB scaffolds are compared. TEHV aim to be designed for humans, but since the ovine model is used to show proof of principle both human and ovine cells were used. Passage 3, 5 and 7 (p3, p5 and p7) human and ovine vascular-derived cells were seeded onto both PGA-P4HB and PCL scaffold strips (n=6 per passage and scaffold material), using fibrin as a cell carrier. After 4 weeks of culture under constrained static conditions, one strip was used for histology. The remaining strips were used for mechanical testing and biochemical assays as indicators for tissue strength and tissue formation, respectively. After 4 weeks, the tissues based on PGA-P4HB showed 50-60% compaction, while PCL-based tissues showed compaction of 0-10%. Cell passage number and species did not influence compaction. Tissue formation was comparable between passage numbers and scaffold materials in ovine while human p5 showed decreased tissue formation in both scaffold materials. Collagen content was increased with decreasing passage numbers in both species and scaffold materials. No differences in cell phenotype between the scaffold materials or cell passage numbers were observed. The Young’s modulus and the ultimate tensile strength of tissues of both species were higher in the lower passage groups of both scaffold groups. This study shows that PCL scaffolds may serve as alternative scaffold material for tissue engineering heart valves with minimal compaction and without compromising on tissue composition and properties. Cells from lower passages showed to improve tissue formation. Reducing cell expansion time will result in faster generation of TEHV, providing a more rapid treatment to patients.
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Fibrin Based Tissue Engineered Heart Valves Featuring the Sinuses of Valsalva
Authors: Petra Mela, Thomas Schermer, Julia Frese, Thomas Schmitz-Rode and Stefan JockenhoevelAbstractThe implementation of the sinuses of Valsalva in both the aortic and the pulmonary position is a crucial step towards the development of functional tissue engineered heart valves with optimal hemodynamic performance and reduced risk of thrombi formation. However, the implementation of these features is not standard in tissue engineered heart valves. In our laboratory we aim at the realization of autologous heart valves starting from materials isolated from the patient (fibrinogen and cells) and shaped into 3D geometries by moulding techniques. We present two new fabrication methods that result in the realization of heart valve scaffolds reproducing the complex geometry of semilunar valves. The first concept consists of a mould in two parts: a ventricular part and a vascular part which contains three removable bulbs representing the sinuses of Valsalva; in the second concept the vascular part features three flexible protrusions shaped as the sinuses of Valsalva which can be collapsed when a vacuum is applied. The moulds were designed with the 3D CAD software Pro/Engineer (PTC, Needham, MA, USA) and manufactured by rapid prototyping. The cell embedded fibrin gel valves were produced by polymerizing a fibrinogen solution in TBS (10 mg/ml) with CaCl2, thrombin and ovine umbilical cord derived fibroblasts (10x106 /ml) suspended in TBS. Afterwards, the obtained construct was placed in a static bioreactor to be cultured on the mould for 14 days in order to avoid cell-mediated tissue contraction before transferring it to a bioreactor for dynamic cultivation of a duration of 2 weeks. To release the fibrin scaffold from the mould after static cultivation the vascular part and subsequently the three removable sinuses of Valsalva were removed (first approach), or the vascular part was collapsed and taken out as one part (second approach). The constructs were successfully released without any tearing with both approaches despite the poor mechanical properties of the fibrin gel. Valved conduits including the sinuses of Valsalva were obtained without the need for suturing any of the parts together. After static and dynamic cultivation the conduits demonstrated good compliance. The tissue development was evaluated by histology (hematoxylin, eosin and immunohistochemistry) hydroxyproline assay and DNA assay. The presence of the sinuses of Valsalva in the aortic and the pulmonary root is fundamental for the correct functioning of semilunar heart valves. The implementation of the sinuses in tissue engineered valves will lead to an improvement of the valve function and an increased durability. Ongoing research focuses on the optimization of the cell source and of the conditioning protocol as to achieve optimal extracellular matrix synthesis and mechanical properties and minimize cell mediated tissue contraction.
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New Biomaterials in Heart Valve Tissue Engineering
AbstractIn situ tissue engineering may lead to a clinically and economically attractive new generation of heart valve replacement therapies, overcoming the limitations of currently used prostheses. Development of novel scaffolds is required for the proposed in situ tissue engineering. These scaffolds must be able to carry a full mechanical load immediately after implantation, and should subsequently provide all biological cues for the recruitment and attachment of specific circulating cells, and should also induce the growth of new tissue. The properties of the scaffold materials should be optimized with respect to mechanical properties and biodegradability characteristics. Synthetic biomaterials are tunable, which enables the creation of tailor made scaffolds suitable for in situ tissue engineering of heart valves. Two different approaches towards scaffold materials have been investigated, based on a biodegradable polyester (PCL or PLLCL), and either a quadruple hydrogen-bonding ureidopyrimidinone (UPy) unit or a bis-urea (BU) moiety. Both were investigated in vivo regarding biocompatibility and degradation kinetics. Thirty rats received the UPy and BU polyester based, disk-shaped implants subcutaneously. To investigate the effects of the implants over time, samples were explanted on day 2, 5, 10, 21 and 84. Disks with surrounding tissue were fixed in 10% neutral buffered formalin. After fixation, explants were dehydrated in graded alcohols and longitudinal sections were embedded in paraffin and stained. From each explant the foreign body response was quantified per high power field. Both the UPy and the BU-based materials proved biocompatible. In the acute phase all investigated biomaterials showed an infiltration of neutrophil granulocytes and mononuclear cells. In the chronic phase encapsulation by fibroblasts took place in all cases. Degradation rates were investigated by gel permeation chromatography (GPC) after 21 and 84 days. Little degradation was observed for the PCL-based polymers over the course of the experiment. The PLLCL-based polymers with a BU moiety, however, showed little at 21 days, but marked degradation at 84 days. Which of the explored materials is best suitable for in situ tissue engineering of heart valves will depend on further experiments, investigating how much time is needed for cell recruitment, adhesion, differentiation and tissue growth.
This research forms part of the Project P1.01 iValve of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. The financial contribution of the Nederlandse Hartstichting is gratefully acknowledged.
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Human Valve Interstitial Cells Demonstrate Transdifferentiation Potential
Authors: Najma Latif, Padmini Sarathchandra, Adrian H Chester and Magdi H, YacoubAbstractAortic stenosis and valvular degeneration is characterized by lipid accumulation, presence of cartilage and calcification. The identity and cellular origins of the cells mediating these effects are unknown. We have investigated the potential of human valve interstitial cells (VICs) to transdifferentiate into osteogenic, adipogenic and chondrogenic-like lineages and examined the presence of resident stem cells. Transdifferentiation potential of VICs was assessed after removal of stem cells. Human aortic heart valves (n=8; mean age 64.7 ± 7.5 years) from patients undergoing transplantation, free from calcification and disease, and were used to isolate VICs. Differentiation was carried out by incubating VICs for 21 days with media containing ascorbate (50µg/ml), dexamethasone (10-8M) and β-glycerophosphate (10mM) for osteogenic differentiation, with ascorbate (50µg/ml), dexamethasone (10-7M) and indomethacin (50µg/ml) for adipogenic differentiation and with insulin transferrin selenium and TGFβ1 (10ng/ml) for chondrogenic differentiation. Immunocytochemistry and fluorescence-activated cell sorting (FACS) were used to assess stem cell populations and removal of sub-populations of VICs. Mesenchymal stem cells were isolated from bone marrow samples obtained from healthy human donors (n=6) and used as positive controls. We analysed the gene expression of some of the Wnt family as potential mediators of transdifferentiation. Incubation of VICs with osteogenic media induced alkaline phosphatase and osteocalcin expression in 34.2 ± 4.6% of VICs, with adipogenic media induced oil red O, SREBP and PPARγ expression in 13.8 ± 5.1% of VICs and with chondrogenic media changed the morphology in 41.7 ± 4.8% of VICs but did not induce collagen type II, type X or aggrecan expression. Cultured VICs expressed CD44 (94.0± 4.4%), CD73 (79.4 ± 15.4%) and CD105 (6.9 ± 2.6%) in common with mesenchymal stem cells. However very low percentages of stem cells were identified in cultured VICs, CD34 (2.8% ± 0.50), CD133 (1.84 ± 0.77%), c-kit (0.72% ± 0.21) and stro-1 (1.55 ± 0.93%). Valve leaflets demonstrated only occasional positive markers for stem cells. Cultured VICs were negative for CD31, CD14, CD45, Tie-2 and flk-1. Removal of these stem cells by FACS demonstrated that purified VICs retained their ability to transdifferentiate into osteogenic and adipogenic lineages to the same degree. Gene expression of the Wnt family showed the expression of Wnt2, Wnt2B, Wnt5B, Wnt 10B. A number of frizzled receptors were detected, FZD2-5 and FZD 6-10 as well as inhibitors DKK 1-3. The population of purified human VICs have the capacity to transdifferentiate into osteogenic, adipogenic and chondrogenic-like lineages. Human valve leaflets and cultures contain a resident stem cell population which can differentiate and thus contribute to valve IC population as well as to pathological phenotypes.
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C-type Natriuretic Peptide Modulates TGF-β1-induced Synthesis of Proteoglycan by VICs
Authors: Mark Blaser, Jung Woo Kwon, Krista L. Sider, Andrea V. Kwong, Kuiru Wei and Craig A. SimmonsAbstractAortic valve disease (AVD) is a cell-mediated pathology marked by unchecked remodeling of the extracellular matrix (ECM). This process is thought to be promoted by increased levels of TGF-β1 in the valve leaflets and the concomitant loss of protective factors such as C-type natriuretic peptide (CNP). Among its many functions, TGF-β1 stimulates the expression of proteoglycan (PG), in part via mitogen activated protein kinase (MAPK)-mediated Smad signalling. We and others have observed the formation of PG-rich lesions in early diseased aortic valves. Here we investigated the relationship between TGF-β1, CNP, and PG in a mouse model of early AVD and in vitro. Male wild-type (WT) C57Bl/6J mice were fed a control diet or BioServ F3282, a high-fat, high-carbohydrate diet (HF/HC) with 58.7% kcal from fat (cholesterol < w/w) for four months (n = 4-6 per group). Longitudinal aortic valve sections from formalin-fixed and paraffin-embedded hearts were stained by Movat’s pentachrome and immunostained for CNP and TGF-β1. Valve interstitial cells (VICs) were isolated from healthy porcine aortic valves and treated in vitro with 5 ng/ml TGF-β1 and/or 1 µM CNP or 10 µM U0126 (inhibitor of MEK, upstream of the MAPK Erk1/2) for up to 6 days. Erk1/2 phosphorylation was measured by Western blot, while PG expression in media collected from these cultures was assayed by Alcian Blue guanidine-HCL solubilisation. Mice on the HF/HC diet for four months became obese, developed mild hypercholesterolemia, and had early AVD, as demonstrated by hemodynamic dysfunction and significant thickening of the leaflets (data shown in another abstract). Thickening was due to PG deposition (11435 ± 7681 vs. 5448 ± 2948 µm2, p < 0.01), not increased collagen content (1729 ± 815 vs. 1771 ± 663 µm2, p = 0.87). TGF-β1 levels were elevated in leaflets of mice on the HF/HC diet, particularly in the region of PG lesions. The PG lesions in HF/HC mice also had lower CNP expression than non-lesion regions. In VICs, TGF-β1 induced a 29% increase of PG expression (p < 0.05), which was significantly reduced by the addition of CNP (p < 0.05). Interactions between TGF-β1 and CNP in PG synthesis were mediated in part via Erk1/2 signaling, as TGF-β1 increased Erk1/2 phosphorylation, CNP abrogated TGF-β1-induced Erk1/2 activation, and blocking Erk1/2 signaling with U0126 significantly reduced PG expression (p = 0.06). These studies demonstrate that in early AVD, thickened leaflets are PG-rich with elevated expression of TGF-β1 and down-regulated CNP. Furthermore, TGF-β1 induces PG expression in VICs, a process which is inhibited by CNP – possibly through Erk1/2. Insight into the mechanisms by which TGF-β1 and CNP regulate the formation of PG lesions helps further our understanding of early disease progression and may aid in identifying novel medical strategies to arrest this disease before a substantive and possibly untreatable burden of calcification occurs.
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The Role Of BMP/Wnt Signalling In Human Heart Valve Calcification
Authors: Paul Riem Vis, Adrian Chester, Najma Latif, Padmini Sarathchandra, Jolanda Kluin and Magdi YacoubAbstractAortic valve calcification is a complex process that is characterised by the expression of bone markers in the valve leaflet. The Wnt pathway and bone morphogenetic proteins (BMPs) have been implicated to play a role in vascular and heart valve calcification. This study examines the regulation of Wnt3a by BMPs and the functional effects mediated by Wnt3a in human valve interstitial cells (VICs). Treatment of VICs by BMPs up-regulated the expression of Wnt3a, ALP, RUNX2, and β-catenin, as was shown by Western Blot analysis. Analysis of human pathological specimens by immunohistochemistry showed an increased level of expression of Wnt3a, Msx2 and β-catenin was localised to peri-calcific regions of the tissue. The concentration-dependent proliferation or differentiation of VICs in response to Wnt3a was measured by the incorporation of [3H]-thymidine and activity of alkaline phosphatase (ALP) respectively. Wnt3a-treated VICs proliferated in a concentration-dependent manner. At higher concentrations, Wnt3a caused a significant increase in ALP activity and expression of RUNX2. Lastly, Western blot experiments showed that BMP2 induced RUNX2 up-regulation was mediated through β-catenin/Wnt-signalling and not Smad-signalling. This study demonstrates the important role of Wnt-signalling in valve calcification in response to BMPs. Wnt3a may induce valve calcification by regulating the proliferation and osteogenic differentiation of cells within the valve. These findings may help to identify new therapeutic.
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Investigation of the Statin Paradox in Different Populations of VICs
Authors: Elyssa L. Monzack, Chloe M. McCoy, Kelsey A. Duxstad and Kristyn S. MastersAbstractWhile numerous clinical studies have examined the effect of HMG-CoA reductase inhibitors (statin drugs) on calcific aortic valve disease (CAVD), their conflicting results have yielded many questions regarding the nature of statin-valve interactions. One step toward better understanding this relationship is to examine the effects of statin treatment on heart valves on a cellular level. Previous work found statin treatment to have a "paradoxical" effect in vitro, decreasing osteoblastic markers in valvular myofibroblasts, while increasing those same markers in osteoblast precursor cells. This finding that statins may be able to selectively induce bone formation only in a cell type that is already prone to mineralization leads to the question of how statin treatment will affect valvular interstitial cells (VICs), a heterogeneous cell population which is capable of differentiating into an osteoblast-like phenotype, termed obVICs. In this study, we set out to determine whether obVICs would respond to statin treatment in the same manner as myofibroblasts, or if obVICs would increase bone marker expression in a manner similar to a bone-derived cell type. This work was also complemented by a gene expression analysis of calcified human valves from individuals who were or were not taking a statin drug. Porcine VICs were cultured in vitro, with or without 1 uM simvastatin, in either control or mineralization medium, where the control medium yields a heterogeneous population that is predominantly myofibroblasts, while the mineralization medium drives VICs toward an obVIC phenotype. Gene expression analysis included multiple myofibroblastic and osteoblastic markers and was conducted daily over an 8-day time course, yielding information about not only expression levels, but also their temporal dynamics. Gene expression profiles were compared between VICs and an osteoblastic cell line (MC3T3-E1) to assess similarities. Myofibroblastic and osteoblastic genes were also analyzed in aortic valves from human patients (+/- statin) undergoing aortic valve replacement surgery. Statin treatment increased osteoblastic gene expression in VICs cultured in mineralization medium (obVICs), but the same effect was not obtained in control medium. This finding suggests that VICs are capable of responding to statin treatment in a manner similar to bone cells, but only when VIC cultures are driven toward an osteoblastic phenotype. The MC3T3-E1 cells also increased osteoblastic gene expression upon statin treatment, although their basal level of osteogenic activity was substantially greater than that found in any of the obVIC cultures. Analysis of human valve data is ongoing. Overall, this study suggests that different subpopulations of VICs exhibit different and temporally dynamic responses to statin treatment, further complicating the ability to predict a clinical effect of statin drugs on CAVD.
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Mitral Valve Interstitial Cells Behaviour Under Hypoxia
AbstractMitral Valve Interstitial Cells (MVICs) are distributed throughout the valve leaflets. It is predicted that some cells exist under hypoxic conditions. Hypoxia is an important stimulus for signalling pathways that affect cell growth differentiation and function. This study examines the effect of various degrees of hypoxia on MVICs growth, survival, morphological and phenotypic behaviour. Porcine MVICs were primarily isolated and incubated under atmospheric control (20% O2), mild hypoxia (5% O2), moderate (2% O2) and severe (0.5% O2) for 1 and 3 days. Cell proliferation and cell death were assessed using biochemical assays. Cell morphology was assessed by immunofluorescence staining. Cells were also stained for phenotypic expression of endothelial, myofibroblastic and smooth muscle markers. After 24 hours incubation at the different O2 concentrations there was no significant difference in cell growth or death. After 3 days incubation cells under atmospheric O2 (150±8%*) and 5% (124±5%), 2% (146±8%*) and 0.5% (161±8%*) all showed increase in cell number compared to start of the experiment (*=P<0.05).However, there was no significant difference between each of the groups. Cell death was significantly reduced under hypoxia (atmospheric O2 (10.65%±1.72) and 5% (8.5%±1.0%), 2% (6.25%±0.24%*) and 0.5% (4.02±0.45%*) O2 (*=P<0.05). Cells were significantly bigger at 3 days under hypoxic conditions but retained the same shape. MVICs continued to express similar levels of myofibroblastic markers αSMA and Vimentin under hypoxic conditions after 3 days but showed weak expression of smooth muscle cell markers. This study serves to define the role of hypoxia in VICs in terms of cell growth, death, morphology and phenotype. These properties further highlight the specialised function of cells that reside in heart valves and have important relevance to heart valve tissue engineering.
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Lysophosphatidylcholine Alters Valvular Interstitial Cell Mineralization
Authors: Dena C. Wiltz, Joel D. Morrisett and K. Jane Grande-AllenAbstractCalcific aortic valve disease (CAVD) is a condition of the heart characterized by thickening and calcification of the aortic valve and can lead to aortic stenosis, narrowing of the aortic valve that can obstruct left ventricular outflow. CAVD is thought to have similarities with atherosclerosis, in which the aortic wall demonstrates thickening due to plaque buildup. A notable similarity seen between CAVD and atherosclerosis is the accumulation of lipids in the tissues. One important chemical component involved in atherosclerosis is lysophosphatidylcholine (LPC), a phospholipid derived from phosphatidylcholine. LPC concentrations have been shown to increase in atherosclerotic conditions, and induce expression of osteogenic factors by vascular smooth muscle cells. The potential for LPC to affect valve cell calcification, however, has not been previously investigated. In addition, calcification of cells from different valves warrants investigation because the aortic valve becomes more bone-like and experiences onset of calcification sooner than the mitral valve during the calcification process. This study investigated the effect of LPC on the propensity for calcification by porcine valve interstitial cells (VICs) from aortic and mitral valves. On day 0 VICs were seeded at a density of 50 000 cells/cm2 in low serum media. On day 1, the media is changed to media containing LPC in concentrations ranging from 0 to 100 µM. The cells are cultured for 8 days and then assessed for mineralization using histological stains (Alizarin Red S for calcium deposition and Von Kossa for phosphate deposition) and biochemical assays (Alkaline phosphatase activity). Significance (p <0.05) was determined using Analysis of Variance followed by Tukey post-hoc testing. Interestingly, mineralization in the VIC cultures was decreased as LPC concentration increased from 0 to 1 µM. At 10 µM, however, an increase in mineralization was observed compared to the 1 µM cultures. VICs in 100 µM LPC media began to detach within 24 hours of LPC media application. Also, VICs from different valves displayed different levels of calcification at each condition. LPC alters mineralization in VIC cultures from both aortic and mitral valves, in a concentration dependent manner. Extremely high concentrations of LPC (at and above 100 µM) can be toxic to VICs. There is a unique behavior of VICs with addition of varying concentrations of LPC, most notably that low concentrations (below 10 µM) actually reduced mineralization. Other factors, such as effects of LPC on VIC proliferation and apoptosis, will be important to investigate in future work. This study demonstrates that LPC affects the mineralization potential of valvular cells in a way that is distinct from vascular cell types.
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Developing a Rat Model of Cardiovascular Calcification to Evaluate Tissue-Engineered Heart Valve Prostheses
AbstractA small animal model to evaluate the in vivo degeneration and calcification of biological versus tissue-engineered cardiovascular prostheses in short time periods is warranted. Our study aims at developing a standardized rat model of accelerated cardiovascular calcification and lipid metabolism disorder. Male Wistar rats (n = 60; 200 – 250g) were fed ad libitum with 5 different regimens of procalcific diet (group 1: +300,000 units/kg vitamin D (VD) +2% cholesterol (CHOL) +1.5% calciumphosphate (PO4); group 2: +150,000 units/kg VD +1% CHOL +0.75% PO4; group 3: +300,000 units/kg VD +2% CHOL; group 4: +300,000 units/kg VD +1.5% PO4; group 5: +2% CHOL + 1.5% PO4; group 6: normal food). After 4, 8 and 12 weeks, animals were euthanized, organs explanted (left ventricular myocardium, aortic valve, ascending aorta, abdominal aorta, kidney and liver) and histology as well as immunohistochemistry conducted. During the study, body weight and chow intake were monitored. Heart function was examined by echocardiography, and blood serum level analyses were conducted at explantation. Unimpaired survival was 100% in all groups. Histology revealed calcification of the aortic valves after 4 weeks, while relevant calcium deposition in the aortic wall was observed only after week 8 (vonKossa staining). Aortic valves of rats with high doses of VD (groups 1, 3 and 4) were significantly more calcified than those of animals with a reduced dose of VD (group 2; p < 0.01) or no VD supplementation (group 5; p < 0.001). In all rats, early calcium deposition was located at the commissures, whereas the aortic sinus walls and especially the valve leaflets were diseased at later time points. Massive calcification was accompanied by chondroid cells and lipid-containing cells (oil red staining). However, animals on a diet with reduced VD or no VD presented a significantly higher amount of chow intake (each with p < 0.01 versus groups 1, 3, 4 and 6), paralleled by significantly larger increase in body as well as heart weight (each with p < 0.001 versus groups 1, 3, 4 and 6). All supplementation regimens resulted in early aortic valve and later aortic wall calcification. High doses of VD intake accelerated the calcium deposition, however, the somatic growth of these rats was impaired. A procalcific diet with moderate doses of VD + CHOL + PO4 seems to be most suitable for a comparative evaluation of calcifying degeneration in native and prosthetic cardiovascular tissues.
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Decellularization Diminishes the Calcifying Degeneration of Aortic Conduit Allografts
AbstractThe present study aimed at developing a small animal transplantation model of accelerated calcifying degeneration, in order to evaluate degenerative in vivo processes in biological heart valves and vascular implants. Male Wistar rats (recipients) with an interventionally induced aortic insufficiency grade II – III (AI; day -14) were fed with a diet containing high-dose vitamin D, cholesterol and calciumphosphate. Aortic conduits of Sprague-Dawley rats (donors) were decellularized according to a detergent-based protocol and infrarenally implanted (day 0) in an end-to-side manner in the recipients (group A; n = 6). Cryopreserved implants served as controls (group B; n = 6). Doppler sonography was conducted at days -14, 0, 28 and 84. Graft explantation, histological and immunohistochemical analyses were performed at days 28 and 84. In all recipients AI grade II – III with subsequent reversed diastolic flow in the abdominal aorta was confirmed. Sonographic competence of the conduit perfusion and overall survival were 100%. After 12 weeks severe calcification of the native aortic media as well as of the aortic conduit implants was observed (vKossa staining), however, in group A diet-induced calcification was significantly lower as compared to group B (p <0.01). Histological evaluation of the conduit implants revealed an intimal hyperplasia, involving α-smooth muscle actin expressing cells, with an increased intima-to-media ratio (p < 0.001) and inflammatory activity (CD3+) in group B versus group A. During the later follow-up, intimal hyperplasia and severe calcification aggravated. After 12 weeks, in opposite to group A explants, all grafts of group B contained Syndecan-3-expressing cells with a chondroid phenotype. Our rapidly calcifying rat transplantation model enables detailed evaluation of native and tissue-engineered aortic conduits, especially in terms of degenerative processes. Compared to cryopreserved grafts, decellularization significantly diminished the calcifying degeneration and intimal hyperplasia of aortic conduit implants. Further work will focus on the characterization of the de novo interstitial repopulation, particularly on the nature of the chondroid cells and their role in graft degeneration.
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Decellularized Heart Valve Prostheses and the Anticalcific Potential of Simvastatin
AbstractDecellularization is a proven approach to decelerate the degenerative processes which lead to the failure of heart valve implants. In order to further delay the calcifying in vivo degeneration of tissue-engineered grafts, antiarteriosclerotic and antiinflammatory substances may be advantageous, and some in vitro reports on HMGCoA reductase inhibitors have shown encouraging results, whereas clinical trials have failed to prove a positive in vivo result. Male Wistar rats (recipients) underwent an interventional generation of aortic insufficiency grade II – III (day -14) and were fed with a procalcific diet of high-dose vitamin D, cholesterol and calciumphosphate, additionally supplemented with simvastatin (group S; n = 6). Identically treated animals fed with the same diet, not supplemented with simvastatin, served as controls (group C; n = 6). Aortic conduits of Sprague-Dawley rats (donors) were decellularized according to a detergent-based protocol and infrarenally implanted (day 0) in an end-to-side manner in the recipients. Echocardiography, doppler sonography of the implant and blood serum analyses were conducted at days -14, 0 and 28. Graft explantation, histological and immunohistochemical analyses as well as quantitative real time PCR were performed after 4 weeks. Acute AI grade II – III with echocardiographically confirmed reversed diastolic flow in the whole aorta caused significant left ventricular (LV) dilatation as well as decrease of LV ejection fraction (p <0.001 at day 28) and resulted in a mortality of 8%. Sonographic competence of the conduit perfusion and overall survival of the transplanted rats were 100%. After 4 weeks of simvastatin treatment, calcification of the implants was significantly lower in group C (p < 0.01), whereas especially the aortic valve and the ascending aorta were strained by a decreased calcium burden (vonKossa staining). Histological evaluation of the conduit implants revealed an intimal hyperplasia, involving α-smooth muscle actin expressing cells, with an increased intima-to-media ratio (p < 0.05) in the aortic walls of group S and in the aortic valves of group C. RNA analysis by quantitative PCR was performed to confirm these results. The present study in a standardized rat transplantation model failed to show an early benefit of the HMGCoA reductase inhibitor simvastatin to diminish the calcification of decellularized aortic conduit implants. Furthermore, existing literature in this field is contradictory, and therefore, further experiments with more detailed analyses and long-term observations are warranted.
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Contribution of Specific Glycosaminoglycans to the Relaxation Properties of the Aortic Valve
Authors: Borghi Borghi, Carubelli Ivan, Adrian H. Chester and Magdi H. YacoubAbstractThe aortic valve (AV) is characterized by a complex mechanical behavior which is closely linked to its structural components. The central layer of the AV is rich in glycosaminoglycans (GAGs) which play an important role in the biomechanics of the AV. In this study the effect of selective GAGs depletion on time dependent mechanical behavior of porcine AV was analyzed. Fresh strips of porcine AV cusps were cut in either in the radial or in the circumferential direction and mounted on a tensile testing system (Bose Electroforce) for mechanical testing. Three groups of valves were treated enzymatically, in order to remover either all the GAGs (group 1), the sulphated GAGs only (group 2) or the non-sulphated GAGs only (group 3). Each group had a control group. Mechanical tests were performed on each strip and stress relaxation kinematics as well as relaxation percentage were compared between treated and untreated specimens. Tensile and stress relaxation tests were performed on the strips under physiological load levels. The reduced stress relaxation function was fitted to the experimental data using a two phase (τ1 and τ2) exponential decay model. Relaxation percentage was significantly lower in group 1 for both circumferential (group 1: 20.58% vs control 28.34%, p = 0.004) and radial strips (group 1: 18.89% vs control 28.85%, p = 0.006). In this group, the early relaxation value (τ1) markedly decreased in the radial direction (group 1: 7.86s vs control: 10.35s, p = 0.0041) while no statistical difference was achieved in the circumferential direction. When looking at selective GAGs depletion, an effect of the hyaluronic acid depletion (group 3) was seen on the excursion of circumferentially oriented strips (group 2: 23.15% vs control: 27.76%, p = 0.049). No major effect was seen comparing the results of group 2 with its control. No effect on t2 was found. Histology confirmed the successful GAGs depletion. The presence of GAGs influences the biomechanics of the AV in terms of time dependent mechanical properties. The presence of hyaluronic acid has a distinctive effect on the relaxation excursion of the cusps while no effect was apparent for sulphated GAGs. These results provide further insight into the relationship between structure and fuction in the AV.
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Characterization of Sex-Related Differences in Valvular Interstitial Cells
Authors: Chloe McCoy, Dylan Q. Nicholas and Kristyn S. MastersAbstractAlthough the pathogenesis of calcific aortic valve disease (CAVD) is not well understood, males have been identified as having a two-fold increased risk for the disease when compared to females. In this study, we examined gene expression profiles in healthy pigs and measured markers of disease in vitro to determine whether the differences in clinical risk between males and females translate into measurable intrinsic differences on the cellular scale. In addition, we also investigated potential sex-related differences in cellular response to TGF-β1, an inflammatory stimulus known to be elevated in calcified human aortic valve explants. mRNA was isolated from three male and three female porcine aortic valves (denuded of endothelial cells) and hybridized to Affymetrix® GeneChip Porcine Genome microarrays. Mean expression values of each probe set in the male samples were compared with those in the female samples and biological processes were analyzed from the dataset for overrepresentation using Gene Ontology term enrichment analysis. From the microarrays there were 183 genes identified as being significantly (fold change>2; P<0.05) different in healthy male versus female aortic valve leaflets. Within this significant gene list there were 298 overrepresented biological processes, several of which are relevant to pathways identified in CAVD pathogenesis. In particular, pathway analysis indicated that cellular proliferation, apoptosis, cell migration, ossification, and extracellular matrix reorganization were all significantly represented in the data set. In vitro culture of male and female porcine valve cells also revealed intrinsic differences between sexes, with male cells exhibiting higher proliferation, apoptosis, and expression of αSMA after five days of culture. When exposed to TGF-β1, male cells grown in serum-free culture were found to be more sensitive to the inflammatory cytokine, with dramatically decreased apoptosis and proliferation compared to a marginal decrease in apoptosis and no change in proliferation in female cells. These data suggest that some sex-related propensity for CAVD may be present on the cellular level in healthy subjects, possibly resulting in a differential response to systemic factors that promote disease onset and progression. These results also offer motivation for further sex-related studies with valve cells to better determine possible genetic contributors and to explore sex-related susceptibilities for valve calcification.
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Influence of Scaffold Structure on Cell Behaviour for Valve Tissue Engineering
AbstractThe success of a tissue engineered heart valve is dependent on developing the right structure, the right interactions between the cells and the right matrix and mechanical force. Different materials have been used as scaffold, however the best processing method have not been established. We have used 2 different type of scaffold: collagen based scaffold and a nanofibrillar scaffold made from poly(ε-caprolactone) (PCL). Different seeding methods have been tested for cell compatibility of these scaffolds with human adipose derived mesenchymal stem cells (hADSCs) and with human telomerase immortalized bone marrow derived stem cells (hTERT). Nanofibrillar scaffolds have been produced by jet-spraying the polymer on a metal grid. Collagen scaffolds were made by freeze drying a 1% bovine collagen solution chemically crosslinked with EDC-NHS. Morphological evaluations of the structures were performed using scanning electron microscopy. Elastic modulus of dry scaffolds (10x6x1 mm, n=5) was measured with a planar biaxial test bench with displacement rate of 0.05 mm/s. Two different type of cells (600000/scaffolds) were seeded using different seeding methods to find the best condition: top seeding for 2 hours and then dynamically cultured using a rotary mixer, directly dynamically seeded for different time period and with different volume (5, 10 and 25 ml). DNA quantification using Hoescht 3258 and DAPI staining were used to evaluate proliferation and penetration inside the scaffold. Immunohistochemistry was used to check collagen production. PCL scaffolds were composed of non woven nanofibers (600 nm average diameter) assembled in a highly open structure. Collagen scaffold showed an interconnected porous structure with average pore size of 100 um. Nanofibrillar scaffolds showed higher elastic modulus compared to collagen (200 KPa compared to 150 KPa). The different cell seeding approaches had an effect on cellular distribution and cell number. With 10 ml of volume cells attached more after 24 hours compared to 5 ml with no further difference compared to 25 ml. Top-seeded matrices resulted in a high cell concentration on the seeded surface while rotary seeding allowed cells to attach on both scaffold sides but in fewer numbers. Regardless of seeding method, cells proliferated extensively (up to 10 and 2-fold DNA increase for hTERT and hADSC respectively ) on both scaffold, but proliferation was up to twice higher within nanofibrillar structures compared to collagen scaffolds. Both cell types were able to populate the entire area of both scaffolds over 10 and 14 culture days for hADSC and hTERT respectively. Both cell type produced their own ECM within the scaffolds as indicated by collagen I positive staining. Jet-sprayed PCL nanofibrillar scaffolds are a promising alternative to collagen scaffolds for cellular infiltration and proliferation.
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Further Refinements in Collagen Mimetic Peptide Scaffolds for Tissue Engineering Heart Valves
AbstractCollagen is the essential protein in the extracellular matrix, which maintains the structural and mechanical integrity of tissues while providing key signals to regulate cell functions. Although animal-based collagens can be used as biomaterial for tissue engineering heart valves, they cause infections and lack flexibility. These limitations have stimulated the exploration of collagen mimetic peptides (CMPs) through a bottom-up approach using computational modeling followed by experiments to enzymatically cross-link the CMPs and produce hydrogels. The X-ray structure of triple-helices of CMP was used in software FIRST and in mutational code to identify its structural stability and hotspots. These data assisted to introduce charged residues by mutations to cross-link and to add binding motif (GFOGER) for integrin in the structure. The helical stability and self-association of the mutated CMP has been validated using molecular dynamics (MD) simulation. Experimentally, the peptide was synthesized by solid phase Fmoc chemistry and characterized by HPLC and mass spectrometry. Enzymatic cross-linking on primary amine and gel formation were obtained by incubating peptide and plasma amine oxydase (PAO) solutions in PBS at 37 and 58 °C. Peptide assembly and aggregation was monitored by turbidity (optical density at 314 nm) and morphology was analysed by transmission electron microscopy (TEM). The modelling analyses indicated the CMP to have the desired structural properties for self-assembly and high affinity towards integrin binding. The modification of the key positions with charged residues increased the possibilities for helical cross-link (gelation). In addition to cell signalling, the charged residues at the cell binding motif could further enhance the inter-helical association of the CMPs. The structural properties of the modelled CMP were reproduced in experimental conditions. Addition of PAO significantly improved turbidity of peptide solutions and lead to hydrogel formation. The peptides assembled in branched fibrillar structures around 25 nm in diameter as it was confirmed by TEM analysis. The proposed peptide promises to show some inherent structural properties of native collagen in silico and in vitro. These properties are required to produce functional scaffolds for tissue engineering heart valves.
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3D Engineered Micro-Tissue Models to Study Cardiovascular (Patho)biology and Regeneration
AbstractEngineered tissue models find their application in studying normal and pathological tissue development and the associated testing of potential therapies. In addition, they provide powerful tools for technology development for regenerative medicine and optimization of regenerative therapies. We have developed a range of ‘humanized’ engineered cardiovascular model systems – consisting of engineered tissue, (hemo)dynamic loading platforms, and readouts of tissue development and mechanical function – for the optimization of in-vitro and in-situ tissue engineering strategies of heart valves and vessels. The model systems can be adapted to simulate either healthy or diseased tissue development or healthy and diseased loading environments (e.g. high blood pressure). A first range of systems consists of cardiovascular tissues (strips or cross-shaped morphology, mm range), engineered from human myofibroblasts seeded on natural (fibrin) or synthetic (PGA) degradable polymer scaffolds, and loaded in series on an adapted Flexcell device to study the mechanobiology of collagen remodeling of engineered tissue. Vital collagen imaging (CNA35) and on-line assessment of structure-function properties indicated that stochastic rather than cyclic loading of the strips resulted in increased collagen formation, organization and tissue strength. A change of loading direction resulted in complete tissue remodeling with collagen re-orientation within 48 hours. Currently, we are using the systems to investigate the pathomorphogenesis of radiation-induced fibrosis of heart valve tissue. A second system consists of a microfluidics-based setup to study circulating cell recruitment, migration and differentiation in small 3D electrospun PCL scaffolds under physiological hemodynamic loading conditions as a model of in-situ regeneration. The system is mounted onto the stage of an inverted confocal microscope to follow cell fate and tissue development in real-time. By changing the architecture, bioactivity and mechanical properties of the scaffold, the effects of these parameters on in-situ tissue formation can be assessed. Circulating cell suspensions of changing composition/activation as well as changing hemodynamic loading conditions will be used to mimic healthy/diseased conditions and to investigate their effects on in-situ tissue regeneration. These studies demonstrate the use of versatile experimental model approaches to provide detailed insight into tissue (patho)morphogenesis, adaptation and regeneration in a real-time and high-throughput fashion.
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Ice-Free-Cryopreservation Attenuates Calcification in Allograft Heart Valves
AbstractThe objective of the study was to attenuate calcification in allogeneic ovine pulmonary heart valves. Six valves of white face sheep were ice-free-cryopreserved (STUDY) in 12.6 mol/L cryoprotectant (4.65, 4.65, and 3.31 mol/L of DMSO, formamide and 1,2-propanediol) and stored at -80°C. 6 control valves were cryopreserved by controlled slow rate freezing in 1.4 mol/L DMSO and stored in vapor-phase nitrogen (CONTROL). After 7 months in vivo explanted valves were processed for histopathology. Gross morphology showed significantly thickened leaflets in the CONTROL group. Histopathology revealed a marked calcification in the leaflet stroma and conduit wall. STUDY valves in contrast demonstrated well preserved ECM structures, no leaflet thickening, inflammation and only neglectable calcification in the conduit wall. Only discrete panus formation was noted migrating from the ventricularis onto the leaflet. Ice-free-cryopreservation enables attenuation of calcification in ovine allografts valves. The observed decreased calcification warrant improved long-term function.
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Sufficient Tissue Engineered Heart Valves: A Question of Cell Source?
Authors: Miriam Weber, Julia Frese, Nima Hatam, Joerg Sachweh, Thomas Schmitz-Rode, Stefanl Jockenhoevel and Petra MelaAbstractA promising approach to solve the problems of currently used heart valve prostheses, e.g. degeneration, need for anticoagulation and risk of endocarditis, is the tissue engineering of heart valves. By using patient derived cells and fibrin as a scaffold for these valves we aim at completely autologous heart valves which have the potential to grow and hence are especially interesting for valve replacement in paediatric surgery. However, a major obstacle on the way to clinical application of tissue engineered heart valves (TEHV) is the cell-mediated tissue contraction which leads to the shrinkage of the valve’s leaflets and thus to its insufficiency. Several groups tested TEHV in the pulmonary position in the sheep model and reported mild to moderate regurgitation already at a short postimplantation time. Our goal was to analyse the influence of the cell source on the sufficiency of TEHV. Different cell sources, among which ovine carotid artery (OCA) and umbilical artery (OUA), were compared on their contractility and contraction of fibrin gels in which the cells were embedded. Cell phenotype was characterized by immunostaining of α-smooth muscle actin (α-SMA) and myosin light-chain kinase (MLCK) as markers for cell contractile activity. For the gel contraction assay, fibrin gels with a cell concentration of 5 × 10^6/ml were moulded in a 24-well plate (n ≥ 3) and their retraction was evaluated over 15 days by measuring the gels' area in relation to their original area. Hydroxyproline content, cell proliferation and burst strength were also determined. Ovine carotid artery cells exhibited a highly contractile myofibroblast phenotype (high α-SMA and MLCK expression), while OUA cells were mostly non-contractile fibroblasts with only few cells expressing α-SMA and MLCK. After 15 days, OCA embedded gels were contracted to 30.6 ± 2.0% of the original size while OUA gels maintained a size of 83.2 ± 3.7% of the initial area. To directly correlate cell contractility and valve sufficiency we moulded fibrin based heart valves using OUA and OCA cells. All valves were conditioned statically for 14 days in the closed-leaflet configuration and successively dynamically in the open-leaflet configuration in bioreactors for 30 days. While the leaflets of TEHV with OCA cells contracted and led to insufficient valves at the end of the conditioning protocol, we were able to produce completely sufficient heart valves after in vitro conditioning using non-contractile OUA cells. In an on-going animal study OUA embedded TEHV, after being seeded with endothelial cells, are implanted in the pulmonary artery in lambs to analyse their growth potential and their hemodynamic performance in vivo. Transesophageal echocardiography showed both in colour Doppler and in cw Doppler mode that after fourteen weeks a first implanted valve was still completely sufficient and showed no sign of cell-mediated tissue contraction.
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Subcutaneous Testing of E-spun PCL Patches Suitable for in Situ Heart Valve Tissue Engineering
AbstractIn situ tissue engineered heart valves yields a new generation of cardiovascular substitutes. The body is used as a bioreactor where it relies on the natural regenerative potential of the body. The shift from the classical way of tissue engineering to in-situ tissue engineering emphasizes the role of the scaffold. The scaffold should be able to capture and preserve cells for tissue formation and it has to maintain valve functionality while tissue is developing. The use of synthetic biomaterials is very attractive. Poly(ε-caprolactone) (PCL) is an important polymer due to its mechanical properties and miscibility with a large range of other polymers. Electrospinning attracted great interest as a production method of biomaterials for in situ tissue engineering. The electrospinning process of PCL offers a nice technique for thin fiber formation to eventually create three dimensional scaffolds with the characteristic three layers, typical for heart valves. The fibers produced with electrospinning provide a similar physical structure as the extra cellular matrix. The space between fibers needs to be large enough for cells to adhere and migrate into the scaffold. Sufficient cellular in growth is needed for tissue formation. In this study cellular in growth was measured in subcutaneously implanted electrospun PCL patches. Furthermore tissue formation and degradation of the polymer is investigated. Thirty healthy male F344 Rats are used. The implants with surrounding tissue were explanted after 2, 5, 10, 21 or 84 days and embedded in paraffin. The explanted tissues were examined using immunohistochemistry (HE,MPO, ED1, α-SMA, and PSR). The electrospun fibers had a diameter of 10 μm. SEM pictures showed controlled void spaces. In the electrospun PCL samples we found a very high cellular infiltration rate after 84 days, mean of 396.819 cells per high power field. First infiltration of mainly neutrophil granulocytes was seen, followed by macrophages. Cells infiltrate throughout the whole sample. After 84 days fibroblast were seen, which were able to produce collagen. Furthermore after 84 days, macrophage giant cells and neo-vessel formation was observed. PCL was degraded in the cytoplasm of the macrophages. Electrospun PCL scaffolds with a fiber diameter of 10 μm, are suitable for cellular in growth and tissue formation. This research forms part of the Project P1.01 iValve of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. The financial contribution of the Nederlandse Hartstichting is gratefully acknowledged.
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Non-Cytotoxic Cross-Linking of Bioactive Porcine Matrices
Authors: Pamela Somers, Filip De Somer, Ria Cornelissen, Hubert Thierens and Guido Van NootenAbstractIncubating a porcine aortic valve matrix with a platelet gel (PG) concentrate creates a bioactive matrix which is loaded with growth factors. These matrices can be repopulated with mesenchymal stem cells. However, these recellularized matrices still elicit a host immune response. Therefore, the aim of this study was to evaluate the cytotoxicity and cross-linking effect of naturally organic compounds such as quercetin, tannic acid, caffeic acid and catechin on these matrices and to investigate the effect of these cross-linkers on the in vitro growth factor release rate. Porcine aortic heart valves were decellularized using a detergent/enzymatic treatment. Cytotoxicty of the cross-linkers was evaluated by cell culture media supplementation of 10, 100, 1000, 5000, 10000 and 20000µg/mL. These concentrations were also used to cross-link the acellular matrices. Mechanical strength of the leaflets was investigated. Also the effect of these cross-linkers on the growth factor release from the PG loaded scaffolds was evaluated by ELISA assays. Results showed that proliferation of porcine mesenchymal stem cells increased significantly with increasing concentrations of quercetin, tannic acid, caffeic acid and catechin. All compounds, except tannic acid, significantly increased mechanical strength of the matrices. Moreover, tensile strength of quercetin cross-linked matrices was comparable to the commercially available 0.625% glutaradehyde fixed valves. Furthermore, cross-linking of the matrices resulted in a decreased burst release of growth factors during the first 4 hours but prolonged the release after 24 hours when compared to non-cross-linked matrices. Natural compounds such as quercetin, caffeic acid and catechin can be used to cross-link porcine aortic valve matrices. Moreover, the in vitro release of growth factors can be prolonged which can be very advantageous in the recellularization of these scaffolds.
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Taurodeoxycholate Overcomes Limitations of Deoxycholate for Xenogenic Cell Removal
AbstractWhen replacement of heart valves is required there is almost no alternative to overcome the shortcomings of the conventional substitutes and the clinical outcomes of recently devised cell-depleted tissue engineered xenogeneic constructs are still controversial. Particularly, osmotic shock- and deoxycholate (DOC)-based acellular preparations that gained approval for use in surgical practice, are reported to have been fully or partly unsuccessful. The formers leading to patient deaths and the others resulting in either a high number of explantations or in successful outcomes at midterm follow-up according to different reports. Experimental evidence obtained in the present investigation indicated that inconsistent clinical outcomes of deoxycholate (DOC)-based heart valve preparations might have been related at least in part to incomplete or variable removal of xenogenic cell material following DOC solubility limitations. Therefore we explored alternatively the efficiency of taurodeoxycholate (TDOC), the highly soluble conjugated form of DOC, associated with Triton X 100 (TRI). Characterization of the resulting acellular scaffold, included shape, volume and mass analysis, quantification of residual xenoantigen alpha-Gal, histology, immunofluorescence, scanning and transmission electron microscopy as well as pulse duplicator testing at systemic pressures. In contrast to previous DOC and combined SDS (sodium dodecyl sulfate)-DOC procedures, adoption of TDOC resulted in complete removal of alpha-Gal xenoantigen, with apparent reduction of laminin and enhanced fibronectin detection by immunofluorescence. Besides cell removal from leaflet, sinus and aortic wall, detailed morphological investigation revealed unconventional aspects of the stromal matrix distribution in native and treated samples. In native samples GAG concentration in spongiosa resulted apparently comparable to that in fibrosa layer while collagen and elastic fibres, respectively, exhibited a peculiar interconnected distribution throughout the valve layers. After TRI-TDOC treatment total leaflet hydration was unchanged while mass, area and thickness decreased. The general hydrodynamic performance of the TRI-TDOC-scaffold well accorded with substantial maintenance of matrix architecture while increased post-treatment gradients and regurgitant volumes correlated with loss of ECM components and partial leaflet retraction. Considering the remarkable cell-removal efficiency and the solubility properties, TDOC is worth of further investigation in the perspective to replace DOC for obtaining xenogenic valve scaffolds free of cell remnants and detergent residues with the aim to restore valvular function in vivo or after dynamic cell culture in vitro.
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AlphaGal Residue in Xenograft Heart Valve Bioprostheses and Tissue Engineered Construct
Authors: Filippo Naso, Alessandro Gandaglia, Giovanni Comacchio, Michele Spina and Gino GerosaAbstractThe glutaraldehyde (GA) fixed heart valve bioprostheses (HVB) fail in the long term due to dystrophic degeneration. To avoid GA treatment, xenogenic tissues have been processed by detergent-based decellularization procedures (DBDP). However a complete immunogeneic tolerance by the host is not granted. Degenerative inflammatory process seems to be triggered by the persistent presence of reactive xenogeneic residual, specifically by the alpha-Gal antigen. Through the use of an ELISA assay we assessed and quantified the content of such xenoantigen in different commercial bioprosthetic heart valves and compare to that present in the native and decellularized tissues used for their manufacture. Four models of pericardial and two of porcine HVBs were investigated for the alpha-Gal content. Untreated porcine aortic leaflets (UPAL) were assessed before and after 3 different detergent-based decellularization procedure: TRICOL (Triton X100 and Sodium Cholate), DOC (Sodium Deoxycholate) and DOC-SDS (Sodium Deoxycholate and Sodium Dodecyl Sulfate). Moreover the total amount of alpha-Gal epitopes in native bovine pericardium (NBP) was determined. All the specimens react with the M86 primary monoclonal antibody and the exposed alpha-Gal epitopes is determined by an indirect ELISA assay. For each sample, the amount of alpha-Gal was expressed as numbers of epitopes *10e11 each 10 mg of wet tissue. The amount of alpha-Gal xenoantigen in pericardial HVB (1.5 ± 0.18 *10e11, n=15) was three and half times less with respect to NBP (5.1 ± 0.21 *10e11, n=9). In a model of porcine HVB the xenoantigen was not detected, in the second one the absolute value (1.37 ± 0.25 *10e11, n=9) was similar to that of the pericardial HVB and half of UPAL (2.5 ± 0.31 *10e11, n=9). DOC and DOC-SDS treatments leave on the tissue the 40% (1.02 ± 0.1 *10e11) of the epitopes originally present in the native cusps. TRICOL has proven to be able to eliminate all the alpha-Gal antigen. HVBs GA-treatment do not prevent the binding of resident alpha-Gal antigens with M86 antibodies. The investigated HVBs exhibited a non negligible amount of reactive epitopes accounting to 29.3% of those exposed by native pericardial tissue and 55% for the porcine one. Probably, the pericardial simpler structure allow a better action of the GA, which is able to ensure a greater, but non complete epitope masking. In one model of porcine HVB the alpha-Gal was not found. Regarding the different DBDPs, the removal of cell components is not a sufficient condition to ensure the elimination of the alpha-Gal epitopes. Up to date the TRICOL seems to be the only method capable of producing an alpha-Gal tissue free.
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The Cellular and Extracellular Matrix Structure of Human Pericardium for Heart Valve Tissue Engineering
AbstractThe objective of our study was to compare the histological structure and cellular organization of autologous human pericardium to that of the human aortic heart valve. Collagen, elastin and glycosaminoglycans are responsible for the mechanical properties of aortic heart valve leaflets. Aortic valve leaflets are composed primarily of collagen representing 50% of total extracellular matrix (ECM). The main collagen types in the aortic heart valve are collagen I (74% of total collagen) and collagen III (24% of total collagen). Elastin represents 13% and is responsible for the elastic properties of the valve leaflet. Collagen has a specific architecture that endows heart valve tissue with the ability to withstand circulatory forces over the course of a lifetime. Its fiber orientation, density and cell associations are very important for this purpose. Normal aortic heart valves were obtained during heart transplantation and compared to autologous human pericardium before and after dynamic conditioning using classical histological assessment, immunohistochemical analysis and confocal microscopy. The architecture of pericardial tissue is very similar to that of the normal aortic heart valve possessing well organized collagen fibers with embedded pericardial interstitial cells (PICs) forming a three dimensional network. Instead of the trilaminar histological organization present in the aortic heart valve, the pericardium possesses one layer whose densely packed collagen bundles closely resemble that of the lamina fibrosa of the native aortic heart valve by confocal microscopy. Elastin fibers are evenly distributed throughout the entire thickness of the pericardium in comparison to the specialized elastin containing layer in the lamina ventricularis of the aortic heart valve. PICs are also evenly distributed throughout the pericardium. In the inner pericardial layer facing the heart these cells have a more spindle-like shape similar to that of valvular interstitial cells (VICs) in the lamina fibrosa, while in the outer part of the pericardium these cells have a more spread-out cytoplasmic morphology interacting with more loosely distributed collagen bundles. Like in the aortic heart valve, PICs show cell-cell interactions in addition to cell-matrix interactions. Our study confirmed similarities in the cellular and ECM organization of human pericardium and the native aortic heart valve. It may be concluded that human autologous pericardium may be a favorable tissue for heart valve replacement. Autologous pericardial tissue may also avoid a negative immune system response that could adversely affect recipient graft uptake and downstream ECM remodeling events.
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Investigation of the Suitability of Decellularised Porcine Pericardium for Mitral Valve Reconstruction
Authors: Lucrezia Morticelli, Daniel Thomas, Eileen Ingham and Sotiris KorossisAbstractThe aim of this study was to investigate the suitability of decellularised porcine pericardium for heterotopic repair of the mitral valve (MV) leaflets, and its potential to regenerate through endogenous cell repopulation in vivo, or in vitro seeding and bioreactor conditioning. Anterior and posterior MV leaflets and pericardia were excised from porcine hearts within. The pericardia were decellularised according to the in-house protocol. Anterior and posterior leaflet, and decellularised and fresh pericardial samples were subjected to histology (H and E, Masson trichrome, Sirius Red, Miller’s elastin, Alcian blue-PAS), immunohistochemistry (collagen type I, III, IV, fibronectin, laminin, and chondroitin sulfate labelling), SEM, and uniaxial tensile testing. Samples were isolated along the radial and circumferential direction (leaflets), and perpendicular and parallel to the collagen fibres (pericardium). Biochemical assays for quantification of the sulphated GAG and collagen content of the tissues were also performed. Contact and extract cytotoxicity testing, and DNA quantification was performed to assess the decellularised pericardia. Histology revealed the trilaminar structure of the pericardium and quadrilaminar structure of the leaflets. Collagen type I and III was found in the fibrosa layers of both pericardium and leaflets, whereas fibronectin and laminin were found throughout the tissues. Decellularisation produced a completely acellular pericardial scaffold, which retained the histoarchitecture of the natural tissue. The biomechanics showed the anterior leaflets being stiffer along the circumferential direction. No significant anisotropy was observed in the biomechanics of the posterior leaflets, or fresh and decellularised pericardium. The anisotropy of the anterior leaflet was attributed to the orientation of the collagen (aligned along the circumferential direction). Biochemistry showed a significant increase in sulphated GAGs between the fresh leaflets and pericardium. No difference was found between the collagen content of the fresh leaflets and the fresh or decellularised pericardium. The decellularised pericardium showed a 99% reduction in DNA and a high loss in the GAG content compared to the fresh pericardium. The study showed that the MV leaflets and pericardium share similar histoarchitectures and comparable biomechanics. The similarity was more pronounced in the case of the posterior leaflet which was more isotropic both in terms of histoarchitecture and biomechanics. Apart from a decreased GAG content, the similarity was also apparent between the leaflets and the pericardial scaffolds. The decellularised pericardium has the potential to deliver the necessary biological and biomechanical cues to seeded or migrating cells, representing a plausible scaffold option for the regeneration of the MV leaflets in vitro or in vivo.
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Integration of Microstructural Architecture of the Mitral Valve into an Anatomically Accurate Finite Element Mesh
AbstractAlthough mitral valve (MV) repair initially restores normal leaflets coaptation and stops MV regurgitation, in long term it can also dramatically change the leaflet geometry and stress distribution that may be in-part responsible for limited repair durability. As shown for other collagenous tissues, such changes in geometry and loading reorganize the fiber architecture. In addition, MV interstitial cells may also respond to the altered stress by reducing biosynthetic function, which would affect the load-bearing capabilities of MV and its long-term durability. Thus, investigating the repair-induced MV stress and the concomitant microstructural alterations is a key step in assessing the repaired valve durability. Finite element models have been widely used for stress analysis of the mitral valve. Most of these models, however, have employed only basic constitutive models and above all ignore the complex microstructure of the MV. In addition, the geometry of the valve is usually simplified. Thus, in this work we developed a method to obtain accurate geometrical model of the ovine MV and quantify its fiber structure for the purposes of developing high fidelity computational meshes of the MV. To obtain an accurate geometry of the MV, microcomputed tomography (micro-CT) was used. The entire heart was scanned via a SIEMENS Inveon CT scanner. Three-dimensional scans were segmented semi-automatically using ScanIP segmentation software. The 3D positional data of the fiducial markers were also obtained via ScanIP masks generated by using gray-scale threshold of the CT scans. The segmented geometry was then converted to finite element meshes using ScanIP mesh free mesh generator scheme. Next, the anterior leaflet was then dissected and prepared for measurements of its fiber alignment. The positional data of each point on the accurate mesh was then projected onto the 3D marker mesh. By using a computational domain, the projected point was mapped back to the 2D flattened surface. In addition to mapping, the current method can be used to estimate the changes in connective tissue structure with deformation. This is done by for each point on the valve surface using the local right Cauchy strain tensor C using an in-plane convective curvilinear coordinate system to convect the local fiber orientation to predict the current fiber alignment. To conclude, a robust technique to quantify and map the fibrous microstructure of the MV anterior leaflet to anatomically accurate 3D MV shape derived from micro-CT imaging was developed. The method provides a framework for development of anatomically and micro-structurally accurate finite element models of MV using our tissue structure-based models. It can also be used as a means to validate predicted changes in fibrous structure due to altered stress following surgical interventions.
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Pressure and Angiotensin II Influence the Mechanical Properties of Aortic Valves
Authors: Valtresa Myles, Jun Liao and James N. WarnockAbstractElevated cyclic pressure and angiotensin II (Ang II) both promote aortic valve collagen synthesis, an early hallmark of aortic sclerosis. In the current study, it was hypothesized that the increased collagen production induced by either elevated pressure or Ang II would increase tissue stiffness. Porcine aortic valve leaflets were randomly assigned to four groups; leaflets in group 1 were treated with 10-6M Ang II, leaflets in group 2 were exposed to 80 or 120 mmHg cyclic pressure, leaflets in group 3 were treated with Ang II and exposed to cyclic pressure and leaflets in group 4 were the control. Biaxial testing was performed after 24 and 48 hours on 10mm x 10mm samples of tissue dissected from the central region of the leaflets. Four fiducial markers arranged in an approximately 4 mm x 4 mm square were placed in the center of the extracted portion of the leaflets to track tissue strain. A membrane tension (force/unit length) was applied along each axis and increased slowly from a pre-stress tension of ~0.5 N/m to a peak tension of 60 N/m. The samples were preconditioned for ten contiguous cycles, following an equibiaxial protocol of TCC:TRR = 60:60 N/m, where TCC and TRR are the tensions applied in the circumferential and radial directions, respectively. Tissue extensibility was characterized by maximum stretch along the circumferential direction (λcc) and maximum stretch along the radial direction (λrr), at an equibiaxial tension of 60 N/m. Leaflet stiffness was greater in the circumferential direction than in the radial direction, which is consistent with previous studies. The peak stretches of native valves were calculated as 1.05±0.02 and 1.40±0.02 in the circumferential and radial directions, respectively. There was no significant difference in the stiffness of native valves compared to those exposed to 0mmHg(-Ang II) or 80mmHg(-Ang II) for 24 or 48 hours. Elevated pressure increased stiffness in both directions after 24 and 48 hours. After 24 and 48 hours Ang II significantly increased stiffness in the radial direction. The combination of Ang II and elevated pressure increased stiffness in radial direction after 24 and 48 hours. Increased stiffness may be due to remodeling of the ECM. Excessive collagen production is known to hinder valve function, eventually resulting in aortic stenosis. In conclusion, the results of the present study demonstrated that both elevated pressure and Ang II play a role in the increased stiffness of aortic valve leaflets.
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Adaptation of the Mitral Valve to Chronic Ischemic Left Ventricular Failure in Swine
Authors: Bryant V. McIver, Samiya Hussain, Vinod Thourani and Muralidhar PadalaAbstractMitral valve leaflets in patients with chronic heart failure are severely stiffened, larger and fibrotic; though the mechanisms underlying such biological remodeling are currently unknown. Chronic tethering of the valve leaflets by the enlarged left ventricle, and the presence of mitral regurgitation (MR), is hypothesized to trigger fibrotic pathways that induce such remodeling. In this study, we sought to develop a large animal model of chronic ischemic left ventricular failure in which moderate leaflet tethering and MR can be repeatedly induced, and the remodeling of the mitral valve can be assessed in vivo using echocardiography. Inferior wall myocardial infarction (IMI) was induced in 16 farm swine (30-35kg) using a percutaneous transfemoral approach. Coronary angiography was performed to selectively identify the marginal branches of the left circumflex artery perfusing the inferior left ventricular (LV) wall and the posterior papillary muscle (PPM), and then these regions were infarcted using a 2 mm balloon catheter and injection of 100% ethyl alcohol into the corresponding branch. Transthoracic 2D echocardiography (TTE) of the left heart was performed pre and post-IMI in all animals, at 4 weeks and at 8 weeks. MR percentage (MR jet/left atrial area), mitral leaflet length and thickness, and mitral annular geometry were measured. Leaflet length and thickness were measured at end diastole to assure measurements were taken in the maximal unloaded state. All animals survived the procedure without complications (0% mortality). At baseline, the swine had mild MR (10.7±8%) which significantly increased to 26.6±9% immediately post-op (p<0.05), declined slightly to 23.8±7.8% at 4 weeks, and increased to 32.2±13.9% at 8 weeks. Mitral annular diameter increased from 2.7±0.5 cm pre-op, to 3.1±0.5 cm post-op (p<0.05), then to 3.8±0.4 cm (p<0.005) and 3.9±0.6 cm (p<0.005), at 4 and 8 weeks, respectively. Posterior leaflet length was 1.8±0.2 cm pre-op, and then stabilized at 1.9±0.2 cm post-op, at 4 weeks, and at 8 weeks. Anterior leaflet length was 2.3±0.3 cm at baseline and increased with the increase in MR, to 2.5±0.4 cm post-op, 2.8±0.5 cm at 4 weeks (p<0.05), and 3.2±0.2 cm at 8 weeks (p<0.001). Posterior leaflet thickness increased from 0.36±0.05 cm at baseline, to 0.37±0.07 cm post-op, and then to 0.4±0.05 cm and 0.41±0.03 cm at 4 weeks and 8 weeks, respectively. Anterior leaflet thickness was 0.4±0.05 cm at baseline and post-operatively, and 0.47±0.03 cm (p<0.01) and 0.45±0.07 cm at 4 weeks and 8 weeks, respectively. Changes in mitral leaflet geometry correspond with changes in the mitral valvular apparatus after IMI in a chronic swine model. As the mitral annulus increases in diameter, significant lengthening is seen from the anterior leaflet, while the posterior leaflet remains relatively static and tethered. The anterior leaflet thickens by 4 weeks, while the posterior leaflet remains stable.
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Valve Calcification on Computed Tomography Can Estimate Aortic Stenosis Severity
AbstractUse of coronary CT angiography for screening of coronary artery disease is being advocated for the general population. Meanwhile, CT is increasingly used in preoperative planning for transcatheter aortic valve replacement (TAVR). Although aortic stenosis (AS) severity can be evaluated by measurement of incidentally found aortic valve calcification on CT, it has not been validated by pathologic specimens and can be confounded by calcification of adjacent structures as well as motion artefact. Micro-computed tomography (microCT) provides ultra-high resolution imaging of small structures to yield excellent estimation of tissue calcification. In this study, excised aortic valves from patients with confirmed AS were used to determine if the amount of calcium on microCT correlated with severity of aortic stenosis. Thirty-five aortic valves excised during surgical valve replacement underwent micro-CT imaging with resolution of 76μm in the axial direction. Amount of calcium was determined by absolute and proportional values of calcium volume. Correlation of calcium volume and preoperative mean aortic valve gradient (MAVG), peak transaortic velocity (Vmax), and aortic valve area (AVA) on echocardiography was evaluated. For the patients who had a preoperative CT scan with acceptable image quality, the amount of valvular calcification was also measured by a well-experienced radiologist using modified Agatston algorithm. Mean amount of calcium across all valves was 603.2±398.5mm3, while mean ratio of calcium volume to total valve volume was 0.36±0.16. Mean aortic valve gradient correlated positively with both calcium volume and ratio (r=0.72, p<0.001). Vmax also positively correlated with calcium volume and ratio (r=0.69 and 0.76 respectively, p<0.001). A logarithmic curvilinear model was best fit to the correlation. Calcium volume of 480mm3 showed sensitivity and specificity of 0.76 and 0.83, respectively for severe AS diagnosis, while calcium ratio of 0.37 yielded sensitivity and specificity of 0.82 and 0.94, respectively. Calcium volume and its proportion to total valve volume were found to be good predictive parameters for severe AS when estimated radiologically. Calcium volume quantification may be a complimentary measure for AS severity evaluation in situations where aortic valve calcification is found incidentally on CT as well as in preoperative assessment of aortic valves for TAVR.
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Mitral Valve Mechanics Following Posterior Leaflet Patch Augmentation
AbstractAttention towards optimization of mitral valve repair methods is increasing. Patch augmentation strategy is used to treat functional ischemic mitral regurgitation (FIMR) and hypertrophic cardiomyopathy. When used to extend the anterior leaflet, the procedure decreases the forces exerted on the secondary chordae. The purpose of this study was therefore to investigate the force balance changes following patch augmentation of the posterior leaflet, with particular attention to the secondary chordae tendineae emanating from the posterior papillary muscle (PPM) in an FIMR simulated valve. Twelve mitral valves were obtained from 80kg pigs. An in vitro test setup simulating the left ventricle was used to hold the valves with a papillary muscle positioning system. Water pressure within the ventricular chamber was regulated manually in order to simulate different static pressures during valve closure. An oval shaped porcine pericardial patch measuring 17x29mm was introduced into the posterior leaflet approximately 2mm from the annulus and extending circumferentially from the middle of P2 to the end of the P3 scallop. In order to simulate a healthy valve, retraction of the patch was performed using sutures, which were then released to simulate patch repair. Data were acquired with and without PPM displacement to simulate the effect from one of the main contributors of FIMR, before and after patch augmentation, giving four simulation scenarios. The PPM was displaced 12mm posteriorly and 5mm apically. Dedicated miniature transducers were used to record the forces exerted on the secondary chordae tendineae. Three-way ANOVA was used to analyze the measurements. The effect of displacing the posterior papillary muscle (p < .010) and implementing patch augmentation (p<.004) are significant and independent of each other. The overall effect of displacing the PPM induced tethering on the secondary chordae tendineae from the PPM to the posterior leaflet resulting in a force increase of 28.2 %. The overall effect of implementing the patch augmentation into the posterior leaflet induced a decrease in force of 24.8 % for the healthy and PPM displaced simulations together. The repairing effect of the patch augmentation is found by comparing the specific scenarios. A 40 % increase is induced by displacing the PPM and a 31 % decrease is found by implementing the patch augmentation, leaving the repaired tethering force a mere 9 % higher than that of the healthy measurements. Posterior leaflet patch augmentation significantly reduced the forces exerted onto the secondary chordae tendineae from the PPM in both healthy and PPM displaced valves. As changes in chordal tension leads to redistribution of the total stress exerted on the valve, patch augmentation may have adverse long term influence on mitral.
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Distensibility of Decellularized and Glutaraldehyde-Preserved Aortic Full Roots as Evaluated by Computed Tomography
AbstractThe aortic root is a dynamic structure and its distensibility is considered an important factor in native and prosthetic heart valve function. Glutaraldehyde-preserved (GLUT) valves have limited durability possibly due to increased tissue stiffening. Decellularized deoxycholic acid-treated (DOA) valves exhibit excellent long-term performance in the pulmonary position in humans possibly due to preservation of valve tissue distensibility. We investigated aortic distensibility in DOA (n=8) and GLUT (n=3) aortic root prostheses in 60 kg pigs 2 weeks after orthotopic implantation. Five pigs served as controls. Using a dual source computed tomography scanner (Somatom Flash, Siemens Medical Solution, Forcheim, Germany) the cross sectional area at the level of Sinus of Valsalva (SoV), sino-tubular junction (STJ), and ascending aorta (AA), respectively, was measured in both diastole and systole. Distensibility was defined as the change in area from diastole (RR 95%) to systole (RR 15%). For assessment of the prosthetic- independent aortic distensibility we performed the same measurements in the descending aorta (AD) in all animals. Data were analyzed using students t-test and reported as a mean±SD. Native aortic distensibility was significant larger at the level of SoV (15.8%±4.9), STJ (46.7%±10.3), and AA (40.6%±9.0), compared with both DOA and GLUT aortic roots (p<0.05). No difference in distensibility between the DOA and GLUT aortic roots were observed: 8.8%±2.4 vs 6.2%±5.5 (SoV), 11.7%±3.0 vs 10.0%±7.1 (STJ), 14.9%±6.1 vs 11.8±1.7 (AA), and 15.6±1.5 vs 15.5±1.8 (DA) , respectively (p>0.05). There was no difference in mean distensibility between the three groups in the decending aorta. This is the first study to evaluate in-vivo distensibility of aortic root prostheses in vivo. Aortic root distensibility is reduced following implantation of DOA or GLUT prosthetic aortic valves. No difference was observed between DOA and GLUT valves. Long-term follow-up is needed in order to verify any changes in bioprosthetic aortic root distensibility over time.
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The Way to a New Generation of Bio-Prostheses
Authors: Thomas Waldow, Katrin Plötze, Matthias König and Klaus MatschkeAbstractDiseases of heart valves often make a prosthetic replacement therapy necessary. Long term goal is the development of artificial leaflet material with defining properties of mechanical prostheses (i.e. durability) with complete biocompatibility without the need for any anticoagulation therapy whatsoever. In order to achieve this aim, objective efforts are being made to use a new material that is still in the process of development. It is a film-like tissue, made of pure carbon in the form of carbon-nanotubes, which are characterized by their excellent mechanical properties (tensile strength 65GPa in comparison to steel with 0.6Gpa). The woven material has also a very small mesh width, which can be influenced by the spinning method. Due to the nature of this material, which is associated with a high surface energy, there is a need for adaptions for the use in vivo according to the requirements. Plasma Enhanced Chemical Vapor Deposition PECVD has proven to be the appropriate method for such a functionalization. It is possible to deposit amorphous hydrocarbon coatings below 60°C and in a thickness range from a few nanometers up to several microns, while at the same time providing a very low Young's modulus that ensures to withstand mechanical stress. In addition, this method allows to influence regional properties e.g. by incorporating silicon into the matrix to prevent adhesion of thrombocytes and/or to add nitrogen to give the ability to endothelial cell growing. The first project step is to evaluate in high resolution and accuracy the parameter characteristic of native valves and prostheses. Therefore the elastic modulus, flexibility, extension, expansion, banding stress, banding elasticity, banding rigidity, reversed banding strength, cantilever load, surface tension, surface structure, surface tension and intensity of surface loading are under investigation. A mix of physical, chemical and visual analysis methods are used. The parameter characteristic of the nanotube material has to match these results. In cases of mismatch a technical adjustment, e.g. during weaving or coating, is possible. At least two big advantages of this new composite material exist: The possibility to produce a compound material with regional different main characteristics by using a two-phase coating process. And on the other hand the abandonment of exogenous substances during the production process and so the material has a distinct advantage over those currently used Teflon fabrics.
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Mechanical Properties of Bioprostheses Leaflets Compared to Human Aortic Valve
Authors: Martins Kalejs, Peteris Stradins, Lacis Lacis, Iveta Ozolanta and Vladimir KasyanovAbstractHeart valve bioprostheses suffer from gradual tissue deterioration, which has a causal link with valve tissue mechanical properties. Limited data on mechanical properties of commercially available bioprostheses comparing them to native human aortic valves (AV) is available. Our objective was to determine the mechanical properties of several contemporary bioprostheses and compare them with native human and porcine aortic valves. Leaflets from 5 unchanged human AV, collected from cadaveric hearts and 5 porcine AV, and from 3 of each kind of bioprostheses - Medtronic Hancock II, Sorin Soprano and Medtronic Freestyle were analysed using uniaxial tensile tests in radial and circumferential directions. Data are presented as means ± standard deviation. In both tested directions there's a shift to the stress axis of stress-strain curve for HancockII prostheses and even more for Soprano prostheses when compared to native human valves. In circumferential direction modulus of elasticity (E) of native human AV is 15.34±3.84MPa, porcine AV - 9.7±1.3MPa, Freestyle - 9.0±3.0MPa, HancockII - 22.5±2.2MPa and Soprano - 29.5±6.0MPa. In radial direction E of native human AV is 1.98±0.15MPa, porcine AV - 1.0±0.2MPa, Freestyle - 0.8±0.3MPa, HancockII - 2.5±0.2MPa and Soprano - 15.8±5.4MPa. Xeno-aortic bioprostheses have a non-linear and anisotropic response to stress in uniaxial tensile tests similar to native AV leaflets. HancockII has gained mechanical strength but lost tissue elasticity compared to native valve tissue. Leaflets of Soprano prostheses are even more rigid and lack pronounced material anisotropy. These differences in mechanical properties may accelerate deterioration of bioprostheses, causing altered stress distribution within valve leaflets. These data provide important information about what mechanical properties future valve substitutes should conform to.
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Polymer Nanofiber Materials Matching the Mechanic Properties of Native Aortic Valve
AbstractPorous electrospun nanofiber materials are very promising as matrices for heart valve tissue engineering. Not only biocompatibility is important for this material but also the mechanical features – it has to be strong enough to withhold the pressure after implantation as well as deformable enough for better distribution of shear stress along its surface. Deformability is also crucial for stimulation of fibre production by fibroblasts on these matrices. Altogether 8 differing density variants of electrospun nanofiber materials from gelatine, polyurethane (PUR), polylactic acid (PLA) and polycaprolactone (PCL) were analysed using uniaxial tensile tests. Data were compared to mechanical properties of porcine aortic valve (AV) leaflets in radial and circumferential directions. Data are presented as means ± standard deviation. In circumferential direction modulus of elasticity (E) of native porcine AV is 9.7±1.3MPa and - 1.0±0.2MPa in radial. Ultimate stress and strain is 44.8±5.9% and 2.3±0.6 MPa in circumferential and 95.6±31.4% and 0.5±0.2MPa in radial direction for native leaflets. Closest of the materials to match the mechanical properties of porcine AV in circumferential direction was PUR with density 6.2 g/sqm showing E of 3.9±0.5 MPa, ultimate stress and strain - 5.3±1.68MPa and 141.8±43.9MPa respectively. Closest to match radial direction was gelatine with density 5.7 g/sqm showing E of 0.64±0.14 MPa, ultimate stress and strain - 0.38±0.05MPa and 82.53±10.20MPa respectively. Native AV leaflets have a non-linear and anisotropic response to stress in uniaxial tensile tests. Hence to model as precisely as possible their mechanical properties we suggest to use a combined material made in a sandwich fashion with layers of gelatine on the outside and PUR in the middle with their fibbers predominantly orientated in perpendicular directions. The other tested materials PLA and PCL either lacked strength to mimic leaflets in circumferential direction or deformability required for the radial direction.
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Functional Mitral Regurgitation Is a Main Determinant Of Adverse Outcome In Patients With Heart Failure Due To Non-Ischemic Dilated Cardiomyopathy.
AbstractIschemic mitral regurgitation has been recently demonstrated to carry important prognostic information in patients with left ventricular dysfunction due to coronary artery disease. There is no information regarding the prognostic role of functional mitral regurgitation in patients with non ischemic dilated cardiomyopathy. Patients with stable heart failure due to non-ischemic dilated cardiomyopathy were prospectively enrolled. All patients underwent a comprehensive echocardiographic assessment. Left ventricular diastolic (LVD) , systolic (LVS) diameters, left atrial diameter (LAD), ejection fraction (EF) and restrictive mitral filling pattern (RMP) was measured. Mitral regurgitant volume (RV) was measured by means of proximal isovelocity surface area method. The end point of the study was death or hospitalization for worsening heart failure. 80 patients (mean age 61±9 years; 82% male) were enrolled. 10 patients reached the end point of the study. At univariate Cox analysis, the echocardiographic variables associated with outcome were: EF (HR 0.84 95% CI 0.75 0.94; p=0.002), RMP (HR 5.2 95% CI 1.4 19.7; p=0.01) and RV (HR 1.046 95% CI 1.02 1.07; p=0.0005), LVS/BSA (HR 1.2 95% CI 1.02 1.4; p=0.03). At multivariate analysis RV remained the only variables independently associated with outcome (p=0.04). Result did not change when LVS/BSA substituted EF in the model. Receiving operator characteristics analysis documented that the area under the curve for RV in identifying patients with adverse outcome was 0.84±0.06 (95% CI 0.74 0.91) and the best cut off value for RV was 28 ml (sensitivity 80% 95% CI 44 97 and specificity 87% 95% CI 77 94). Patients with RV<28 had a survival rate of 95% after 6 years from the index echocardiogram compared with 22% in those with RV> 28 (longrank 23; p<0.0001). In patients with non-ischemic dilated cardiomyopathy, RV was a main predictor of death or hospitalization for worsening heart failure.
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Prevalence of Severe Aortic Stenosis With Low Flow in Patients With and Without Left Ventricular Dysfunction. A Consecutive Echocardiographic Population
AbstractPatients with aortic stenosis (AS) may have a severely reduced aortic valve area (AVA) and a paradoxically low mean gradient (MG). Although this condition is well known in presence of left ventricular (LV) dysfunction, it has recently been observed that it can be associated with normal ejection fraction (EF) as well. Since the prevalence of this condition with respect to LV function is not well defined, we aimed to evaluate the distribution of patients with severely reduced valve area and low MG in a group of patients regardless of EF over a set period of time. We retrospectively identified consecutive patients with severe AS (defined as aortic valve area <0.6 cmq/mq) from our echo data-base. Low MG was defined as < 40 mmHg. Left ventricular systolic dysfunction was considered as EF< 50%. 167 patients with AVA <0.6 cmq/mq formed the study population. 94 (56%) patients were characterized by high MG and 73 (44%) by low MG. Among patients with low MG, 38 were characterized by normal EF and 35 by reduced EF. Differences among groups are shown in Table 1. In this echocardiographic series of consecutive patients, the prevalence of low MG despite severely reduced AVA was high. The distribution of low MG was similar in presence of reduced and preserved EF (21 and 23% of the overall population respectively).
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